Publications by authors named "Aloysius J Klingelhutz"

58 Publications

Human Keratinocyte Response to Superantigens.

mSphere 2020 10 7;5(5). Epub 2020 Oct 7.

Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

and are significant human pathogens, causing infections at multiple body sites, including across the skin. Both are organisms that cause human diseases and secrete superantigens, including toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxins (SEs), and streptococcal pyrogenic exotoxins (SPEs). On the skin, human keratinocytes represent the first cell type to encounter these superantigens. We employed transcriptome sequencing (RNA-seq) to evaluate the human primary keratinocyte response to both TSST-1 and staphylococcal enterotoxin B (SEB) in triplicate analyses. Both superantigens caused large numbers of genes to be up- and downregulated. The genes that exhibited 2-fold differential gene expression compared to vehicle-treated cells, whether up- or downregulated, totaled 5,773 for TSST-1 and 4,320 for SEB. Of these, 4,482 were significantly upregulated by exposure of keratinocytes to TSST-1, whereas 1,291 were downregulated. For SEB, expression levels of 3,785 genes were upregulated, whereas those of 535 were downregulated. There was the expected high overlap in both upregulation (3,412 genes) and downregulation (400 genes). Significantly upregulated genes included those associated with chemokine production, with the possibility of stimulation of inflammation. We also tested an immortalized human keratinocyte line, from a different donor, for chemokine response to four superantigens. TSST-1 and SEB caused production of interleukin-8 (IL-8), MIP-3α, and IL-33. SPEA and SPEC were evaluated for stimulation of expression of IL-8 as a representative chemokine; both stimulated production of IL-8. and are common human pathogens, causing infections that include the skin. Both pathogens produce a family of secreted toxins called superantigens, which have been shown to be important in human diseases. The first cell types encountered by superantigens on skin are keratinocytes. Our studies demonstrated, that the human keratinocyte pathway, among other pathways, responds to superantigens with production of chemokines, setting off inflammation. This inflammatory response may be harmful, facilitating opening of the skin barrier.
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http://dx.doi.org/10.1128/mSphere.00803-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568652PMC
October 2020

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

PLoS One 2020 30;15(7):e0236741. Epub 2020 Jul 30.

Department of Microbiology and Immunology, Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, IA, United States of America.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236741PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392206PMC
September 2020

Human Adipocyte Conditioned Medium Promotes In Vitro Fibroblast Conversion to Myofibroblasts.

Sci Rep 2020 06 24;10(1):10286. Epub 2020 Jun 24.

Roy J. Carver Department of Biomedical Engineering, College of Engineering, University of Iowa, Iowa City, IA, USA.

Adipocytes and adipose tissue derived cells have been investigated for their potential to contribute to the wound healing process. However, the details of how these cells interact with other essential cell types, such as myofibroblasts/fibroblasts, remain unclear. Using a novel in-vitro 3D human adipocyte/pre-adipocyte spheroid model, we investigated whether adipocytes and their precursors (pre-adipocytes) secrete factors that affect human dermal fibroblast behavior. We found that both adipocyte and pre-adipocyte conditioned medium induced the migration of fibroblasts, but only adipocyte conditioned medium induced fibroblast differentiation into a highly contractile, collagen producing myofibroblast phenotype. Furthermore, adipocyte mediated myofibroblast induction occurred through a TGF-β independent mechanism. Our findings contribute to a better understanding on the involvement of adipose tissue in wound healing, and may help to uncover and develop fat-related wound healing treatments.
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http://dx.doi.org/10.1038/s41598-020-67175-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314785PMC
June 2020

Serovars Drive Differential Production of Proinflammatory Cytokines and Chemokines Depending on the Type of Cell Infected.

Front Cell Infect Microbiol 2019 26;9:399. Epub 2019 Nov 26.

Department of Microbiology and Immunology, University of Iowa Carver College of Medicine, Iowa City, IA, United States.

serovars A-C infect conjunctival epithelial cells and untreated infection can lead to blindness. D-K infect genital tract epithelial cells resulting in pelvic inflammatory disease, ectopic pregnancy, and sterility while L1-L3 infect epithelial cells and macrophages, causing an invasive infection. Despite some strains of sharing high nucleotide sequence similarity, the bacterial and host factors that govern tissue and cellular tropism remain largely unknown. Following introduction of via intercourse, epithelial cells of the vagina, foreskin, and ectocervix are exposed to large numbers of the pathogen, yet their response to infection and the dynamics of chlamydial growth in these cells has not been well-characterized compared to growth in more permissive cell types that harbor . We compared intracellular replication and inclusion development of representative serovars in immortalized human conjunctival epithelial, urogenital epithelial, PMA stimulated THP-1 (macrophages), and HeLa cells. We demonstrate that urogenital epithelial cells of the vagina, ectocervix, and foreskin restrict replication of serovar A while promoting robust replication and inclusion development of serovar D and L2. Macrophages restrict serovars D and A while L2 proliferates in these cells. Furthermore, we show that GM-CSF, RANTES, GROα, IL-1α, IL-1β, IP-10, IL-8, and IL-18 are produced in a cell-type and serovar-specific manner. Collectively we have established a series of human cell lines that represent some of the first cell types to encounter following exposure and show that differential production of key cytokines early during infection could regulate serovar-host cell specificity.
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http://dx.doi.org/10.3389/fcimb.2019.00399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988789PMC
August 2020

PCB126 blocks the thermogenic beiging response of adipocytes.

Environ Sci Pollut Res Int 2020 Mar 12;27(9):8897-8904. Epub 2019 Nov 12.

Department of Microbiology and Immunology, University of Iowa, 3-612 BSB, 51 Newton Road, Iowa City, IA, 52242, USA.

Subcutaneous white adipose tissue is capable of becoming thermogenic in a process that is referred to as "beiging." Beiging is associated with activation of the uncoupling protein, UCP1, and is known to be important for preventing adipose hypertrophy and development of insulin resistance. Polychlorinated biphenyls (PCBs) accumulate in fat, and it is hypothesized that disruption of adipogenesis and adipocyte function by PCBs may be causative in the development of obesity and diabetes. We developed immortal human subcutaneous preadipocytes that, when differentiated, are capable of beiging. Preadipocytes that were treated with polychlorinated biphenyl congener 126 (PCB126), followed by differentiation, were suppressed for their ability to activate UCP1 upon β-adrenergic stimulation with norepinephrine (NE), demonstrating a block in the beiging response. Treatment of preadipocytes with another known endogenous AhR agonist, indoxyl sulfate (IS), followed by differentiation also blocked the NE-stimulated upregulation of UCP1. Knockdown of the aryl hydrocarbon receptor (AhR) caused the preadipocytes to be refractory to PCB126 and IS effects. The chemical AhR antagonist, CH223191, was effective at preventing the effects of PCB126 but not IS, indicating AhR ligand specificity of CH223191. Repression of NE-induced UCP1 upregulation was also observed when already-differentiated mature adipocytes were treated with PCB126 but not IS. These results indicate that exposure of preadipocytes to endogenous (IS) or exogenous (PCB126) AhR agonists is effective at blocking them from becoming functional adipocytes that are capable of the beiging response. Mature adipocytes may have differential responses. This finding suggests a mechanism by which dioxin-like PCBs such as PCB126 could lead to disruption in energy homeostasis, potentially leading to obesity and diabetes.
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http://dx.doi.org/10.1007/s11356-019-06663-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098842PMC
March 2020

Staphylococcal Superantigens Stimulate Epithelial Cells through CD40 To Produce Chemokines.

mBio 2019 03 19;10(2). Epub 2019 Mar 19.

Department of Pediatrics, National Jewish Health, Denver, Colorado, USA.

Mucosal and skin tissues form barriers to infection by most bacterial pathogens. causes diseases across these barriers in part dependent on the proinflammatory properties of superantigens. We showed, through use of a CRISPR-Cas9 CD40 knockout, that the superantigens toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxins (SEs) B and C stimulated chemokine production from human vaginal epithelial cells (HVECs) through human CD40. This response was enhanced by addition of antibodies against CD40 through an unknown mechanism. TSST-1 was better able to stimulate chemokine (IL-8 and MIP-3α) production by HVECs than SEB and SEC, suggesting this is the reason for TSST-1's exclusive association with menstrual TSS. A mutant of TSST-1, K121A, caused TSS in a rabbit model when administered vaginally but not intravenously, emphasizing the importance of the local vaginal environment. Collectively, our data suggested that superantigens facilitate infections by disruption of mucosal barriers through their binding to CD40, with subsequent expression of chemokines. The chemokines facilitate TSS and possibly other epithelial conditions after attraction of the adaptive immune system to the local environment. Menstrual toxic shock syndrome (TSS) is a serious infectious disease associated with vaginal colonization by producing the exotoxin TSS toxin 1 (TSST-1). We show that menstrual TSS occurs after TSST-1 interaction with an immune costimulatory molecule called CD40 on the surface of vaginal epithelial cells. Other related toxins, where the entire family is called the superantigen family, bind to CD40, but not with a high-enough apparent affinity to cause TSS; thus, TSST-1 is the only exotoxin superantigen associated. Once the epithelial cells become activated by TSST-1, they produce soluble molecules referred to as chemokines, which in turn facilitate TSST-1 activation of T lymphocytes and macrophages to cause the symptoms of TSS. Identification of small-molecule inhibitors of the interaction of TSST-1 with CD40 may be useful so that they may serve as additives to medical devices, such as tampons and menstrual cups, to reduce the incidence of menstrual TSS.
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http://dx.doi.org/10.1128/mBio.00214-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426597PMC
March 2019

Scaffold-free generation of uniform adipose spheroids for metabolism research and drug discovery.

Sci Rep 2018 01 11;8(1):523. Epub 2018 Jan 11.

University of Iowa Fraternal Order of Eagles Diabetes Research Center, 169 Newton Rd, Iowa City, IA, 52242, USA.

Adipose tissue dysfunction is critical to the development of type II diabetes and other metabolic diseases. While monolayer cell culture has been useful for studying fat biology, 2D culture often does not reflect the complexity of fat tissue. Animal models are also problematic in that they are expensive, time consuming, and may not completely recapitulate human biology because of species variation. To address these problems, we have developed a scaffold-free method to generate 3D adipose spheroids from primary or immortal human or mouse pre-adipocytes. Pre-adipocytes self-organize into spheroids in hanging drops and upon transfer to low attachment plates, can be maintained in long-term cultures. Upon exposure to differentiation cues, the cells mature into adipocytes, accumulating large lipid droplets that expand with time. The 3D spheroids express and secrete higher levels of adiponectin compared to 2D culture and respond to stress, either culture-related or toxin-associated, by secreting pro-inflammatory adipokines. In addition, 3D spheroids derived from brown adipose tissue (BAT) retain expression of BAT markers better than 2D cultures derived from the same tissue. Thus, this model can be used to study both the maturation of pre-adipocytes or the function of mature adipocytes in a 3D culture environment.
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http://dx.doi.org/10.1038/s41598-017-19024-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765134PMC
January 2018

The Superantigen Toxic Shock Syndrome Toxin 1 Alters Human Aortic Endothelial Cell Function.

Infect Immun 2018 03 20;86(3). Epub 2018 Feb 20.

Department of Microbiology and Immunology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

infective endocarditis (IE) is a fast-progressing and tissue-destructive infection of the cardiac endothelium. The superantigens (SAgs) toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin C (SEC), and the toxins encoded by the enterotoxin gene cluster () play a novel and essential role in the etiology of IE. Recent studies indicate that SAgs act at the infection site to cause tissue pathology and promote vegetation growth. The underlying mechanism of SAg involvement has not been clearly defined. In SAg-mediated responses, immune cell priming is considered a primary triggering event leading to endothelial cell activation and altered function. Utilizing immortalized human aortic endothelial cells (iHAECs), we demonstrated that TSST-1 directly activates iHAECs, as documented by upregulation of vascular and intercellular adhesion molecules (VCAM-1 and ICAM-1). TSST-1-mediated activation results in increased monolayer permeability and defects in vascular reendothelialization. Yet stimulation of iHAECs with TSST-1 fails to induce interleukin-8 (IL-8) and IL-6 production. Furthermore, simultaneous stimulation of iHAECs with TSST-1 and lipopolysaccharide (LPS) inhibits LPS-mediated IL-8 and IL-6 secretion, even after pretreatment with either of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β. IL-8 suppression is not mediated by TSST-1 binding to its canonical receptor major histocompatibility complex class II (MHC-II), supporting current evidence for a nonhematopoietic interacting site on SAgs. Together, the data suggest that TSST-1 differentially regulates cell-bound and secreted markers of endothelial cell activation that may result in dysregulated innate immune responses during IE. Endothelial changes resulting from the action of SAgs can therefore directly contribute to the aggressive nature of IE and development of life-threatening complications.
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http://dx.doi.org/10.1128/IAI.00848-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820935PMC
March 2018

A delayed proinflammatory response of human preadipocytes to PCB126 is dependent on the aryl hydrocarbon receptor.

Environ Sci Pollut Res Int 2018 Jun 11;25(17):16481-16492. Epub 2017 Jul 11.

Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA.

Inflammation in adipose tissue is recognized as a causative factor in the development of type II diabetes. Adipocyte hypertrophy as well as bacterial and environmental factors have been implicated in causing inflammation in mature adipocytes. Exposure to persistent organic pollutants such as polychlorinated biphenyls (PCBs) has been associated with the development of type II diabetes. We show here that PCB126, a dioxin-like PCB, activates a robust proinflammatory state in fat cell precursors (preadipocytes). The response was found to be dependent on aryl hydrocarbon receptor (AhR) activation, although induction of the response was delayed compared to upregulation of CYP1A1, a classic AhR-responsive gene. Treatment of preadipocytes with a nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) inhibitor partially attenuated the PCB126-induced inflammatory response and partly, but not completely, ameliorated disruption of adipogenesis caused by PCB126. Our results indicate a role for PCB126 in mediating an inflammatory response through AhR in preadipocytes that interferes with adipogenesis.
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http://dx.doi.org/10.1007/s11356-017-9676-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764822PMC
June 2018

Staphylococcal β-Toxin Modulates Human Aortic Endothelial Cell and Platelet Function through Sphingomyelinase and Biofilm Ligase Activities.

mBio 2017 03 21;8(2). Epub 2017 Mar 21.

Department of Microbiology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE). IE is an endovascular infection of native and prosthetic valves and the lining of the heart; it is characterized by the formation of cauliflower-like "vegetations" composed of fibrin, platelets, other host factors, bacteria, and bacterial products. β-Toxin is an virulence factor that contributes to the microorganism's ability to cause IE. This cytolysin has two enzymatic activities: sphingomyelinase (SMase) and biofilm ligase. Although both activities have functions in a rabbit model of IE, the mechanism(s) by which β-toxin directly affects human cells and is involved in the infectious process has not been elucidated. Here, we compared the effects of purified recombinant wild-type β-toxin, SMase-deficient β-toxin (H289N), and biofilm ligase-deficient β-toxin (H162A and/or D163A) on human aortic endothelial cells (HAECs) and platelets. β-Toxin was cytotoxic to HAECs and inhibited the production of interleukin 8 (IL-8) from these cells by both SMase and biofilm ligase activities. β-Toxin altered HAEC surface expression of CD40 and vascular cell adhesion molecule 1 (VCAM-1). HAECs treated with β-toxin displayed granular membrane morphology not seen in treatment with the SMase-deficient mutant. The altered morphology resulted in two possibly separable activities, cell rounding and redistribution of cell membranes into granules, which were not the result of endosome production from the Golgi apparatus or lysosomes. β-Toxin directly aggregated rabbit platelets via SMase activity. Each year there are up to 100,000 cases of infective endocarditis (IE) in the United States. is the most common pathogen in patients with health care-associated IE and the leading cause of community-associated IE in the developed world. Multiple clonal group strains as defined by the Centers for Disease Control and Prevention, particularly USA200 and other clones encoding β-toxin, are highly associated with IE. Considering the strong association and established contribution of β-toxin in animal models of IE, determining how β-toxin directly affects human cell types, including endothelial cells and platelets, is important. In this study, we demonstrate that β-toxin functions to modulate endothelial cells and platelets by both toxin sphingomyelinase and biofilm ligase activities. Our data suggest that these activities modulate inflammation and increase infection severity.
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http://dx.doi.org/10.1128/mBio.00273-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362035PMC
March 2017

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

COPD 2017 Apr 27;14(2):228-237. Epub 2016 Dec 27.

a Department of Medicine , University of Pittsburgh , Pittsburgh , PA , USA.

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.
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http://dx.doi.org/10.1080/15412555.2016.1262340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362315PMC
April 2017

Diminished Phosphorylation of CREB Is a Key Event in the Dysregulation of Gluconeogenesis and Glycogenolysis in PCB126 Hepatotoxicity.

Chem Res Toxicol 2016 09 23;29(9):1504-9. Epub 2016 Aug 23.

Interdisciplinary Graduate Program in Human Toxicology and Department of Occupational and Environmental Health, University of Iowa , Iowa City, Iowa 52242, United States.

The dioxin-like PCB126 elicits toxicity in various target organs. In rat liver, an alteration in the transcript levels of several genes involved in glucose and fatty acid metabolism provides insights into the origin of its hepatotoxicity. To explore the mechanisms, male Sprague-Dawley rats, fed an AIN-93G diet, were injected with PCB126 (1 or 5 μmol/kg) or corn oil and euthanized after 2 weeks. PCB126 significantly decreased serum glucose levels and the transcript levels of genes of many gluconeogenic and glycogenolytic enzymes under the transcriptional control of a nuclear transcription factor, cAMP response element-binding protein (CREB). As a novel finding, we show that PCB126 significantly decreases CREB phosphorylation, which is important for regulating both gluconeogenesis and fatty acid oxidation in the liver and explains CREB's integrative effects on both carbohydrate and lipid metabolism in PCB126 toxicity.
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http://dx.doi.org/10.1021/acs.chemrestox.6b00172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298789PMC
September 2016

Suppression of Resting Metabolism by the Angiotensin AT2 Receptor.

Cell Rep 2016 08 28;16(6):1548-1560. Epub 2016 Jul 28.

Department of Pharmacology, University of Iowa, Iowa City, IA 52242, USA; Fraternal Order of Eagles' Diabetes Research Center, University of Iowa, Iowa City, IA 52242, USA; Obesity Research and Education Initiative, University of Iowa, Iowa City, IA 52242, USA; François M. Abboud Cardiovascular Research Center, University of Iowa, Iowa City, IA 52242, USA; Center for Hypertension Research, University of Iowa, Iowa City, IA 52242, USA. Electronic address:

Activation of the brain renin-angiotensin system (RAS) stimulates energy expenditure through increasing of the resting metabolic rate (RMR), and this effect requires simultaneous suppression of the circulating and/or adipose RAS. To identify the mechanism by which the peripheral RAS opposes RMR control by the brain RAS, we examined mice with transgenic activation of the brain RAS (sRA mice). sRA mice exhibit increased RMR through increased energy flux in the inguinal adipose tissue, and this effect is attenuated by angiotensin II type 2 receptor (AT2) activation. AT2 activation in inguinal adipocytes opposes norepinephrine-induced uncoupling protein-1 (UCP1) production and aspects of cellular respiration, but not lipolysis. AT2 activation also opposes inguinal adipocyte function and differentiation responses to epidermal growth factor (EGF). These results highlight a major, multifaceted role for AT2 within inguinal adipocytes in the control of RMR. The AT2 receptor may therefore contribute to body fat distribution and adipose depot-specific effects upon cardio-metabolic health.
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http://dx.doi.org/10.1016/j.celrep.2016.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981564PMC
August 2016

Does Staphylococcus aureus have a role in the development of Type 2 diabetes mellitus?

Future Microbiol 2015 6;10(10):1549-52. Epub 2015 Oct 6.

Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.

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http://dx.doi.org/10.2217/fmb.15.95DOI Listing
May 2016

PCB126-Induced Disruption in Gluconeogenesis and Fatty Acid Oxidation Precedes Fatty Liver in Male Rats.

Toxicol Sci 2016 Jan 22;149(1):98-110. Epub 2015 Sep 22.

*Interdisciplinary Graduate Program in Human Toxicology; Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa City, Iowa;

3,3',4,4',5-Pentachlorobiphenyl (PCB126), a dioxin-like polychlorinated biphenyl (PCB) and a potent aryl hydrocarbon receptor (AhR) agonist, is implicated in the disruption of both carbohydrate and lipid metabolism which ultimately leads to wasting disorders, metabolic disease, and nonalcoholic fatty liver disease. However, the mechanisms are unclear. Because liver is the target organ for PCB toxicity and responsible for metabolic homeostasis, we hypothesized that early disruption of glucose and lipid homeostasis contributes to later manifestations such as hepatic steatosis. To test this hypothesis, groups of male Sprague Dawley rats, fed on AIN-93G diet, were injected (intraperitoneal.) with a single bolus of PCB126 (5 µmol/kg) at various time intervals between 9 h and 12 days prior to euthanasia. An early decrease in serum glucose and a gradual decrease in serum triglycerides were observed over time. Liver lipid accumulation was most severe at 6 and 12 days of exposure. Transcript levels of cytosolic phosphoenol-pyruvate carboxykinase (Pepck-c/Pck1) and glucose transporter (Glut2/Slc2a2) involved in gluconeogenesis and hepatic glucose transport were time-dependently downregulated between 9 h and 12 days of PCB126 exposure. Additionally, transcript levels of Pparα, and its targets acyl-CoA oxidase (Acox1) and hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2), were also downregulated, indicating changes in peroxisomal fatty acid oxidation and ketogenesis. In a separate animal study, we found that the measured changes in the transcript levels of Pepck-c, Glut2, Pparα, Acox1, and Hmgcs2 were also dose dependent. Furthermore, PCB126-induced effects on Pepck-c were demonstrated to be AhR dependent in rat H4IIE hepatocytes. These results indicate that PCB126-induced wasting and steatosis are preceded initially by (1) decreased serum glucose caused by decreased hepatic glucose production, followed by (2) decreased peroxisomal fatty acid oxidation.
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http://dx.doi.org/10.1093/toxsci/kfv215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731404PMC
January 2016

Novel Staphylococcus aureus Secreted Protein Alters Keratinocyte Proliferation and Elicits a Proinflammatory Response In Vitro and In Vivo.

Biochemistry 2015 Aug 28;54(31):4855-62. Epub 2015 Jul 28.

§University of Colorado, Denver, Anschutz Medical Campus, Aurora, Colorado 80045, United States.

Staphylococcus aureus is a leading cause of surgical site infections that results in increased hospital stays due to the development of chronic wounds. Little is known about factors involved in S. aureus' ability to prevent wounds from healing. We discovered a novel secreted protein produced by a surgical site isolate of S. aureus that prevents keratinocyte proliferation. The protein has a molecular weight of 15.7 kDa and an isoelectric point of 8.9. The cloned and purified protein has cytotoxic and proinflammatory properties, as shown in vitro and in vivo. Potent biological effects on keratinocytes and rabbit skin suggest that this protein may play an important role in preventing re-epithelialization. Its lack of homology to known exotoxins suggests that this protein is novel, and this observation is likely to open a new field of research in S. aureus exotoxins. Due to its cytotoxic activities, we call this new protein ε-cytotoxin.
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http://dx.doi.org/10.1021/acs.biochem.5b00523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912018PMC
August 2015

Chronic superantigen exposure induces systemic inflammation, elevated bloodstream endotoxin, and abnormal glucose tolerance in rabbits: possible role in diabetes.

mBio 2015 Feb 24;6(2):e02554. Epub 2015 Feb 24.

Department of Microbiology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA

Unlabelled: Excessive weight and obesity are associated with the development of diabetes mellitus type 2 (DMII) in humans. They also pose high risks of Staphylococcus aureus colonization and overt infections. S. aureus causes a wide range of severe illnesses in both healthy and immunocompromised individuals. Among S. aureus virulence factors, superantigens are essential for pathogenicity. In this study, we show that rabbits that are chronically exposed to S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1) experience impaired glucose tolerance, systemic inflammation, and elevated endotoxin levels in the bloodstream, all of which are common findings in DMII. Additionally, such DMII-associated findings are also seen through effects of TSST-1 on isolated adipocytes. Collectively, our findings suggest that chronic exposure to S. aureus superantigens facilitates the development of DMII, which may lead to therapeutic targeting of S. aureus and its superantigens.

Importance: Obesity has a strong correlation with type 2 diabetes, in which fatty tissue, containing adipocytes, contributes to the development of the illness through altered metabolism and chronic inflammation. The human microbiome changes in persons with obesity and type 2 diabetes, including increases in Staphylococcus aureus colonization and overt infections. While the microbiome is essential for human wellness, there is little understanding of the role of microbes in obesity or the development of diabetes. Here, we demonstrate that the S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1), an essential exotoxin in pathogenesis, induces inflammation, lipolysis, and insulin resistance in adipocytes both in vitro and in vivo. Chronic stimulation of rabbits with TSST-1 results in impaired systemic glucose tolerance, the hallmark finding in type 2 diabetes in humans, suggesting a role of S. aureus and its superantigens in the progression to type 2 diabetes.
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http://dx.doi.org/10.1128/mBio.02554-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358007PMC
February 2015

PCB126 inhibits adipogenesis of human preadipocytes.

Toxicol In Vitro 2015 Feb 7;29(1):132-41. Epub 2014 Oct 7.

Department of Microbiology, The University of Iowa, Iowa City, IA 52242, United States. Electronic address:

Emerging evidence indicates that persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs), are involved in the development of diabetes. Dysfunctional adipocytes play a significant role in initiating insulin resistance. Preadipocytes make up a large portion of adipose tissue and are necessary for the generation of functional mature adipocytes through adipogenesis. PCB126 is a dioxin-like PCB and a potent aryl hydrocarbon receptor (AhR) agonist. We hypothesized that PCB126 may be involved in the development of diabetes through disruption of adipogenesis. Using a newly developed human preadipocyte cell line called NPAD (Normal PreADipocytes), we found that exposure of preadipocytes to PCB126 resulted in significant reduction in their subsequent ability to fully differentiate into adipocytes, more so than when the cells were exposed to PCB126 during differentiation. Reduction in differentiation by PCB126 was associated with downregulation of transcript levels of a key adipocyte transcription factor, PPARγ, and late adipocyte differentiation genes. An AhR antagonist, CH223191, blocked this effect. These studies indicate that preadipocytes are particularly sensitive to the effects of PCB126 and suggest that AhR activation inhibits PPARγ transcription and subsequent adipogenesis. Our results validate the NPAD cell line as a useful model for studying the effects of POPs on adipogenesis.
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http://dx.doi.org/10.1016/j.tiv.2014.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250299PMC
February 2015

RABL6A promotes G1-S phase progression and pancreatic neuroendocrine tumor cell proliferation in an Rb1-dependent manner.

Cancer Res 2014 Nov 1;74(22):6661-70. Epub 2014 Oct 1.

Department of Pharmacology, University of Iowa, Iowa City, Iowa. Molecular and Cellular Biology Graduate Program, University of Iowa, Iowa City, Iowa. Medical Scientist Training Program, University of Iowa, Iowa City, Iowa. The Holden Comprehensive Cancer Center, University of Iowa, Iowa City, Iowa. Department of Pathology, College of Medicine, University of Iowa, Iowa City, Iowa.

Mechanisms of neuroendocrine tumor (NET) proliferation are poorly understood, and therapies that effectively control NET progression and metastatic disease are limited. We found amplification of a putative oncogene, RABL6A, in primary human pancreatic NETs (PNET) that correlated with high-level RABL6A protein expression. Consistent with those results, stable silencing of RABL6A in cultured BON-1 PNET cells revealed that it is essential for their proliferation and survival. Cells lacking RABL6A predominantly arrested in G1 phase with a moderate mitotic block. Pathway analysis of microarray data suggested activation of the p53 and retinoblastoma (Rb1) tumor-suppressor pathways in the arrested cells. Loss of p53 had no effect on the RABL6A knockdown phenotype, indicating that RABL6A functions independent of p53 in this setting. By comparison, Rb1 inactivation partially restored G1 to S phase progression in RABL6A-knockdown cells, although it was insufficient to override the mitotic arrest and cell death caused by RABL6A loss. Thus, RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET proliferation and development. This work identifies RABL6A as a novel negative regulator of Rb1 that is essential for PNET proliferation and survival. We suggest RABL6A is a new potential biomarker and target for anticancer therapy in PNET patients.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-3742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233153PMC
November 2014

Acrolein-exposed normal human lung fibroblasts in vitro: cellular senescence, enhanced telomere erosion, and degradation of Werner's syndrome protein.

Environ Health Perspect 2014 Sep 18;122(9):955-62. Epub 2014 Apr 18.

Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of New Mexico and New Mexico VA Health Care System, Albuquerque, New Mexico, USA.

Background: Acrolein is a ubiquitous environmental hazard to human health. Acrolein has been reported to activate the DNA damage response and induce apoptosis. However, little is known about the effects of acrolein on cellular senescence.

Objectives: We examined whether acrolein induces cellular senescence in cultured normal human lung fibroblasts (NHLF).

Methods: We cultured NHLF in the presence or absence of acrolein and determined the effects of acrolein on cell proliferative capacity, senescence-associated β-galactosidase activity, the known senescence-inducing pathways (e.g., p53, p21), and telomere length.

Results: We found that acrolein induced cellular senescence by increasing both p53 and p21. The knockdown of p53 mediated by small interfering RNA (siRNA) attenuated acrolein-induced cellular senescence. Acrolein decreased Werner's syndrome protein (WRN), a member of the RecQ helicase family involved in DNA repair and telomere maintenance. Acrolein-induced down-regulation of WRN protein was rescued by p53 knockdown or proteasome inhibition. Finally, we found that acrolein accelerated p53-mediated telomere shortening.

Conclusions: These results suggest that acrolein induces p53-mediated cellular senescence accompanied by enhanced telomere attrition and WRN protein down-regulation.
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http://dx.doi.org/10.1289/ehp.1306911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154210PMC
September 2014

Staphylococcal superantigens stimulate immortalized human adipocytes to produce chemokines.

PLoS One 2013 30;8(10):e77988. Epub 2013 Oct 30.

Department of Microbiology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, United States of America.

Background: Human adipocytes may have significant functions in wound healing and the development of diabetes through production of pro-inflammatory cytokines after stimulation by gram-negative bacterial endotoxin. Diabetic foot ulcers are most often associated with staphylococcal infections. Adipocyte responses in the area of the wound may play a role in persistence and pathology. We studied the effect of staphylococcal superantigens (SAgs) on immortalized human adipocytes, alone and in the presence of bacterial endotoxin or staphylococcal α-toxin.

Methodology/principal Findings: Primary non-diabetic and diabetic human preadipocytes were immortalized by the reverse transcriptase component of telomerase (TERT) and the E6/E7 genes of human papillomavirus. The immortal cells were demonstrated to have properties of non-immortalized pre-adipocytes and could be differentiated into mature and functional adipocytes. Differentiated adipocytes exposed to staphylococcal SAgs produced robust levels of cytokines IL-6 and IL-8, but there were no significant differences in levels between the non-diabetic and diabetic cells. Cytokine production was increased by co-incubation of adipocytes with SAgs and endotoxin together. In contrast, α-toxin alone was cytotoxic at high concentrations, but, at sub-cytotoxic doses, did not stimulate production of IL-6 and IL-8.

Conclusions/significance: Endotoxin has been proposed to contribute to diabetes through enhanced insulin resistance after chronic exposure and stimulation of adipocytes to produce cytokines. Our data indicate staphylococcal SAgs TSST-1 and SEB alone and in combination with bacterial endotoxin also stimulate adipocytes to produce cytokines and thus may contribute to the inflammatory response found in chronic diabetic ulcers and in the systemic inflammation that is associated with the development and persistence of diabetes. The immortal human pre-adipocytes reported here will be useful for studies to understand further the mechanism by which toxins are involved in wound healing and the development and clinical manifestations of obesity and diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077988PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813495PMC
September 2014

Identification of RNA aptamers that internalize into HPV-16 E6/E7 transformed tonsillar epithelial cells.

Virology 2013 Nov 8;446(1-2):325-33. Epub 2013 Sep 8.

Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.

Human papillomavirus type 16 (HPV-16) associated oropharyngeal cancers are on a significant increase and better therapeutic strategies are needed. The HPV-16 oncogenes E6 and E7 are expressed in HPV-associated cancers and are able to transform human tonsillar epithelial cells (HTECs). We used cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to select for RNA aptamers that entered into HPV-16 E6/E7-HTECs. After 12 rounds of cell-SELEX, a pool of aptamers was obtained that had significantly greater internalization capacity (~5-fold) into E6/E7-HTECs as compared to primary HTECs or fibroblasts. Analysis of individual aptamers from the pool indicated variable internalization into E6/E7-HTECs (1-8-fold as compared to a negative control). Most of the individual aptamers internalized into E6/E7 and primary HTECs with similar efficiency, while one aptamer exhibited ~3-fold better internalization into E6/E7-HTECs. Aptamers that internalize into cells may be useful for delivering therapeutic agents to HPV-16 associated malignancies.
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http://dx.doi.org/10.1016/j.virol.2013.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812236PMC
November 2013

Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers.

Pharmaceuticals (Basel) 2013 Mar;6(3):295-319

Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method () and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs .
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http://dx.doi.org/10.3390/ph6030295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3722562PMC
March 2013

Enhanced radiation sensitivity in HPV-positive head and neck cancer.

Cancer Res 2013 Aug 7;73(15):4791-800. Epub 2013 Jun 7.

Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, 3107 WIMR, 1111 Highland Avenue, Madison, WI 53705, USA.

Patients with human papillomavirus (HPV+)-associated head and neck cancer (HNC) show significantly improved survival outcome compared with those with HPV-negative (HPV-) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV- HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV-: 0.59 vs. HPV+: 0.22; P < 0.0001), corresponding with a prolonged G2-M cell-cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%; P = 0.002). A genome-wide microarray was used to compare gene expression 24 hours following radiation between HPV+ and HPV- cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6-fold increase in TP53 (P < 0.0001). Using immortalized human tonsillar epithelial (HTE) cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV- HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-0587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732540PMC
August 2013

Papillomavirus E6 oncoproteins.

Virology 2013 Oct 24;445(1-2):115-37. Epub 2013 May 24.

Department of Pathology, University of Virginia, Charlottesville, VA 22901, USA. Electronic address:

Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on these proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression.
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http://dx.doi.org/10.1016/j.virol.2013.04.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3783570PMC
October 2013

Dyskeratosis Congenita Dermal Fibroblasts are Defective in Supporting the Clonogenic Growth of Epidermal Keratinocytes.

Aging Dis 2012 Dec 12;3(6):427-37. Epub 2012 Oct 12.

Department of Pediatrics, University of Iowa, Iowa City, IA, USA.

Telomere shortening is associated with cellular senescence and aging. Dyskeratosis congenita (DC) is a premature aging syndrome caused by mutations in genes for telomerase components or telomere proteins. DC patients have very short telomeres and exhibit aging-associated pathologies including epidermal abnormalities and bone marrow failure. Here, we show that DC skin fibroblasts are defective in their ability to support the clonogenic growth of epidermal keratinocytes. Conditioned media transfer experiments demonstrated that this defect was largely due to lack of a factor or factors secreted from the DC fibroblasts. Compared to early passage normal fibroblasts, DC fibroblasts express significantly lower transcript levels of several genes that code for secreted proteins, including Insulin-like Growth Factor 1 (IGF1) and Hepatocyte Growth Factor (HGF). Aged normal fibroblasts with short telomeres also had reduced levels of IGF1 and HGF, similar to early passage DC fibroblasts. Knockdown of IGF1 or HGF in normal fibroblasts caused a reduction in the capacity of conditioned media from these fibroblasts to support keratinocyte clonogenic growth. Surprisingly, reconstitution of telomerase in DC fibroblasts did not significantly increase transcript levels of IGF1 or HGF or substantially increase the ability of the fibroblasts to support keratinocyte growth, indicating that the gene expression defect is not readily reversible. Our results suggest that telomere shortening in dermal fibroblasts leads to reduction in expression of genes such as IGF1 and HGF and that this may cause a defect in supporting normal epidermal proliferation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522509PMC
December 2012

Cellular transformation by human papillomaviruses: lessons learned by comparing high- and low-risk viruses.

Virology 2012 Mar 27;424(2):77-98. Epub 2012 Jan 27.

Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA.

The oncogenic potential of papillomaviruses (PVs) has been appreciated since the 1930s yet the mechanisms of virally-mediated cellular transformation are still being revealed. Reasons for this include: a) the oncoproteins are multifunctional, b) there is an ever-growing list of cellular interacting proteins, c) more than one cellular protein may bind to a given region of the oncoprotein, and d) there is only limited information on the proteins encoded by the corresponding non-oncogenic PVs. The perspective of this review will be to contrast the activities of the viral E6 and E7 proteins encoded by the oncogenic human PVs (termed high-risk HPVs) to those encoded by their non-oncogenic counterparts (termed low-risk HPVs) in an attempt to sort out viral life cycle-related functions from oncogenic functions. The review will emphasize lessons learned from the cell culture studies of the HPVs causing mucosal/genital tract cancers.
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http://dx.doi.org/10.1016/j.virol.2011.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703738PMC
March 2012

ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers.

PLoS One 2012 18;7(1):e30447. Epub 2012 Jan 18.

Cancer Biology Research Center, Sanford Research/University of South Dakota, Sioux Falls, South Dakota, United States of America.

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030447PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261204PMC
July 2012

Low-temperature electrospun silk scaffold for in vitro mucosal modeling.

J Biomed Mater Res A 2012 Mar 11;100(3):757-67. Epub 2012 Jan 11.

VCU Philips Institute, Virginia Commonwealth University, Richmond, Virginia 23298, USA.

Electrospinning is often used to create scaffolding as a biomimetic of the extracellular matrix of tissues. A frequent limitation of this technique for three-dimensional tissue modeling is poor cell infiltration throughout the void volume of scaffolds. Here, we generated low-temperature electrospun silk scaffolds and compared these with conventional electrospun silk scaffolds in terms of mechanical properties, void volume, cell infiltration, cell viability, and potential to support mucosal models under three-dimensional culture conditions. Low-temperature electrospun silk scaffolds supported fibroblast attachment and infiltration throughout the volume of the scaffolds, while conventional electrospun scaffolds exhibited limited cell infiltration with fibroblasts attaching exclusively to the seeding surface of the scaffolds. The porosity of low-temperature electrospun scaffolds was 93% compared with 88% of conventional electrospun silk scaffolds. Uniaxial tensile testing showed a 3.5-fold reduction in strength of low-temperature electrospun silk compared with the conventional in terms of peak stress and modulus but no significant change in strain at break. Mucosal modeling with fibroblast-keratinocyte or fibroblast-carcinoma cocultures showed similar results, with cell infiltration occurring only in low-temperature electrospun scaffolds. Cell viability was confirmed using live/dead staining after 21 days in culture. Furthermore, low-temperature electrospun silk scaffolds were able to support keratinocyte differentiation, as judged by involucrin immunoreactivity. The low-temperature electrospun silk scaffold that we have developed eliminates the limitation of electrospun silk scaffolds in terms of cell infiltration and, therefore, can potentially be used for a wide range of tissue engineering purposes ranging from in vitro tissue modeling to in vivo tissue regeneration purposes.
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http://dx.doi.org/10.1002/jbm.a.33288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266471PMC
March 2012

Therapeutic opportunities: telomere maintenance in inducible pluripotent stem cells.

Mutat Res 2012 Feb 13;730(1-2):98-105. Epub 2011 May 13.

Department of Microbiology, University of Iowa, IA, USA.

It has been demonstrated that exogenous expression of a combination of transcription factors can reprogram differentiated cells such as fibroblasts and keratinocytes into what have been termed induced pluripotent stem (iPS) cells. These iPS cells are capable of differentiating into all the tissue lineages when placed in the right environment and, in the case of mouse cells, can generate chimeric mice and be transmitted through the germline. Safer and more efficient methods of reprogramming are rapidly being developed. Clearly, iPS cells present a number of exciting possibilities, including disease modeling and therapy. A major question is whether the nuclei of iPS cells are truly rejuvenated or whether they might retain some of the marks of aging from the cells from which they were derived. One measure of cellular aging is the telomere. In this regard, recent studies have demonstrated that telomeres in iPS cells may be rejuvenated. They are not only elongated by reactivated telomerase but they are also epigenetically modified to be similar but not identical to embryonic stem cells. Upon differentiation, the derivative cells turn down telomerase, the telomeres begin to shorten again, and the telomeres and the genome are returned to an epigenetic state that is similar to normal differentiated somatic cells. While these preliminary telomere findings are promising, the overall genomic integrity of reprogrammed cells may still be problematic and further studies are needed to examine the safety and feasibility of using iPS cells in regenerative medicine applications.
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http://dx.doi.org/10.1016/j.mrfmmm.2011.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3179558PMC
February 2012