Medical Cell Biology Commons^™

Intermediary Role Of Lung Alveolar Type 1 Cells In Epithelial Repair Upon Sendai Virus Infection, Belinda J Hernandez, Margo P Cain, Anne M Lynch, Jose R Flores, Michael J Tuvim, Burton F Dickey, Jichao Chen

Faculty, Staff and Student Publications

The lung epithelium forms the first barrier against respiratory pathogens and noxious chemicals; however, little is known about how more than 90% of this barrier, made of AT1 (alveolar type 1) cells, responds to injury. Using the Sendai virus to model natural infection in mice, we find evidence that AT1 cells have an intermediary role by persisting in areas depleted of AT2 cells, upregulating IFN responsive genes, and receding from invading airway cells. Sendai virus infection mobilizes airway cells to form alveolar SOX2+ (Sry-box 2+) clusters without differentiating into AT1 or AT2 cells. Large AT2 cell-depleted areas remain covered by …

Hematopoietic-Mesenchymal Signals Regulate The Properties Of Mesenchymal Stem Cells, Sanshiro Kanazawa, Hiroyuki Okada, Dan Riu, Yo Mabuchi, Chihiro Akazawa, Junichi Iwata, Kazuto Hoshi, Atsuhiko Hikita

Faculty, Staff and Student Publications

It is well known that the properties of hematopoietic stem/progenitor cells (HSCs), such as their self-renewal ability and multipotency, are maintained through interactions with mesenchymal stem/stromal cells (MSCs). MSCs are rare cells that are present in the bone marrow and are useful for clinical applications due to their functional ability. To obtain the necessary number of cells, MSCs must be cultured to expand, but this causes a remarkable decrease in stem cell properties, such as multipotency and proliferation ability. In this study, we show that the c-Mpl signal, which is related to the maintenance of hematopoietic stem cells, has an …

Alveolar Socket Healing In 5-Lipoxygenase Knockout Aged Female Mice Treated Or Not With High Dose Of Zoledronic Acid, Ramez H Mahmoud, Claudia C Biguetti, Gustavo B Simionato, Isabela C Custódio, Raquel B P Silva, Marco A H Duarte, Leonardo P Faverani, Edilson Ervolino, Walid D Fakhouri, Mariza A Matsumoto

Faculty, Staff and Student Publications

This study investigated the role 5-lypoxigenase (5-LO) on alveolar socket healing in aged female mice treated with zoledronic acid (ZL). Forty 129/Sv female mice (64-68 weeks old), 20 wild type (WT) and 20 5-LO knockout (5LOKO) were equally distributed according to ZL treatment: WT Control, WT ZL, 5LOKO Control, and 5LOKO ZL. ZL groups were treated with an intraperitoneal injection of 250 µg/Kg of ZL, while controls were treated with saline. Treatments were administered once a week, starting four weeks before surgery for tooth extraction and until 7 and 21 days post-surgery. Mice were euthanized for a comprehensive microscopic analysis …

Decorin-Evoked Paternally Expressed Gene 3 (Peg3) Is An Upstream Regulator Of The Transcription Factor Eb (Tfeb) In Endothelial Cell Autophagy., Thomas Neill, Catherine Sharpe, Rick T. Owens, Renato V. Iozzo

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Macroautophagy is a fundamental and evolutionarily conserved catabolic process that eradicates damaged and aging macromolecules and organelles in eukaryotic cells. Decorin, an archetypical small leucine-rich proteoglycan, initiates a protracted autophagic program downstream of VEGF receptor 2 (VEGFR2) signaling that requires paternally expressed gene 3 (PEG3). We have discovered that PEG3 is an upstream transcriptional regulator of transcription factor EB (TFEB), a master transcription factor of lysosomal biogenesis, for decorin-evoked endothelial cell autophagy. We found a functional requirement of PEG3 for TFEB transcriptional induction and nuclear translocation in human umbilical vein endothelial and PAER2 cells. Mechanistically, inhibiting VEGFR2 or AMP-activated protein …

Natural Killer (Nk) Cells And Their Involvement In Different Types Of Cancer. Current Status Of Clinical Research, Isadora Zaharescu, Adina D. Moldovan, Cristiana Tanase

Journal of Mind and Medical Sciences

Natural killer cells are the main agents of innate immunity. Since 1970, various studies have repeatedly confirmed their involvement in decreasing local tumor growth and also decreasing the risk of metastasis, due to their cytotoxic effects and also through the release of immunostimulatory cytokines such as IFN-gamma. In the 1990s, several studies demonstrated the existence of certain inhibiting and stimulating receptors of these cells, leading to the concept of “induced self”, thus explaining why tumors with MHC-1 are destroyed and autologous cells without it are saved out. Recognition and destruction of tumor cells by the NK cells are the result …

Influence Of Environmental Exposure On Human Epigenetic Regulation, C. J. Marsit

Dartmouth Scholarship

Environmental toxicants can alter epigenetic regulatory features such as DNA methylation and microRNA expression. As the sensitivity of epigenomic regulatory features may be greatest during the in utero period, when critical windows are narrow, and when epigenomic profiles are being set, this review will highlight research focused on that period. I will focus on work in human populations, where the impact of environmental toxicants in utero, including cigarette smoke and toxic trace metals such as arsenic, mercury and manganese, on genome-wide, gene-specific DNA methylation has been assessed. In particular, arsenic is highlighted, as this metalloid has been the focus …

Inpp4b Suppresses Prostate Cancer Cell Invasion, Myles C. Hodgson, Elena I. Deryugina, Egla Suarez, Sandra M. Lopez, Dong Lin, Hui Xue, Ivan P. Gorlov

Dartmouth Scholarship

INPP4B and PTEN dual specificity phosphatases are frequently lost during progression of prostate cancer to metastatic disease. We and others have previously shown that loss of INPP4B expression correlates with poor prognosis in multiple malignancies and with metastatic spread in prostate cancer.

We demonstrate that de novo expression of INPP4B in highly invasive human prostate carcinoma PC-3 cells suppresses their invasion both in vitro and in vivo. Using global gene expression analysis, we found that INPP4B regulates a number of genes associated with cell adhesion, the extracellular matrix, and the cytoskeleton. Importantly, de novo expressed INPP4B suppressed the proinflammatory chemokine …

Enhanced Pancreatic Beta-Cells Proliferation And Functionality, Hanan Abdulaziz Alismail

Graduate Theses and Dissertations

Biologically functional beta-cells proliferate at an extremely low rate with limited turnover capacity. This cellular property hinders cell-based therapy for clinical applications. Many attempts have been made to develop techniques that allow large quantities of production of clinically relevant islet β-cells in vitro. A line of studies demonstrates that functional beta-cells can proliferate under certain circumstances, providing hope for generating and expanding these cells in vitro and transplanting them into the recipient. In this study, we showed that a membrane substrate offers a better niche for beta cell proliferation and insulin secretion. Mouse beta cells were grown on a tissue …

Mammalian Alteration/Deficiency In Activation 3 (Ada3) Is Essential For Embryonic Development And Cell Cycle Progression., Shakur Mohibi, Channabasavaiah B. Gurumurthy, Alo Nag, Jun Wang, Sameer Mirza, Yousaf Mian, Meghan Quinn, Bryan J. Katafiasz, James D. Eudy, Sanjit Pandey, Chittibabu Guda, Mayumi Naramura, Hamid Band, Vimla Band

Journal Articles: Genetics, Cell Biology & Anatomy

Ada3 protein is an essential component of histone acetyl transferase containing coactivator complexes conserved from yeast to human. We show here that germline deletion of Ada3 in mouse is embryonic lethal, and adenovirus-Cre mediated conditional deletion of Ada3 in Ada3(FL/FL) mouse embryonic fibroblasts leads to a severe proliferation defect which was rescued by ectopic expression of human Ada3. A delay in G(1) to S phase of cell cycle was also seen that was due to accumulation of Cdk inhibitor p27 which was an indirect effect of c-myc gene transcription control by Ada3. We further showed that this defect could be …

Orai1 Deficiency Leads To Heart Failure And Skeletal Myopathy In Zebrafish., Mirko Völkers, Nima Dolatabadi, Natalie Gude, Patrick Most, Mark A Sussman, David Hassel

Center for Translational Medicine Faculty Papers

Mutations in the store-operated Ca²⁺ entry pore protein ORAI1 have been reported to cause myopathies in human patients but the mechanism involved is not known. Cardiomyocytes express ORAI1 but its role in heart function is also unknown. Using reverse genetics in zebrafish, we demonstrated that inactivation of the highly conserved zebrafish orthologue of ORAI1 resulted in severe heart failure, reduced ventricular systolic function, bradycardia and skeletal muscle weakness. Electron microscopy of Orai1-deficient myocytes revealed progressive skeletal muscle instability with loss of myofiber integrity and ultrastructural abnormalities of the z-disc in both skeletal and cardiac muscle. Isolated Orai1-deficient cardiomyocytes showed loss …

The Interplay Between Nf-Kappab And E2f1 Coordinately Regulates Inflammation And Metabolism In Human Cardiac Cells., Xavier Palomer, David Álvarez-Guardia, Mercy M Davidson, Tung O Chan, Arthur M Feldman, Manuel Vázquez-Carrera

Department of Medicine Faculty Papers

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, …

S-Glutathionylation Activates Stim1 And Alters Mitochondrial Homeostasis., Brian J Hawkins, Krishna M Irrinki, Karthik Mallilankaraman, Yu-Chin Lien, Youjun Wang, Cunnigaiper D Bhanumathy, Ramasamy Subbiah, Michael F Ritchie, Jonathan Soboloff, Yoshihiro Baba, Tomohiro Kurosaki, Suresh K Joseph, Donald L Gill, Muniswamy Madesh

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca(2+) signaling. However, precisely how oxidants influence Ca(2+) signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca(2+) mobilization from an oscillatory to a sustained elevated pattern via calcium release-activated calcium (CRAC)-mediated capacitive Ca(2+) entry, and stromal interaction molecule 1 (STIM1)- and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca(2+) entry alters mitochondrial Ca(2+) handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to …

Differential Impact Of Tumor Suppressor Pathways On Dna Damage Response And Therapy-Induced Transformation In A Mouse Primary Cell Model., A Kathleen Mcclendon, Jeffry L Dean, Adam Ertel, Erik S Knudsen

Department of Cancer Biology Faculty Papers

The RB and p53 tumor suppressors are mediators of DNA damage response, and compound inactivation of RB and p53 is a common occurrence in human cancers. Surprisingly, their cooperation in DNA damage signaling in relation to tumorigenesis and therapeutic response remains enigmatic. In the context of individuals with heritable retinoblastoma, there is a predilection for secondary tumor development, which has been associated with the use of radiation-therapy to treat the primary tumor. Furthermore, while germline mutations of the p53 gene are critical drivers for cancer predisposition syndromes, it is postulated that extrinsic stresses play a major role in promoting varying …

Role Of Mammalian Ecdysoneless In Cell Cycle Regulation., Jun Hyun Kim, Channabasavaiah B. Gurumurthy, Mayumi Naramura, Ying Zhang, Andrew T. Dudley, Lynn Doglio, Hamid Band, Vimla Band

Journal Articles: Genetics, Cell Biology & Anatomy

The Ecdysoneless (Ecd) protein is required for cell-autonomous roles in development and oogenesis in Drosophila, but the function of its evolutionarily conserved mammalian orthologs is not clear. To study the cellular function of Ecd in mammalian cells, we generated Ecd(lox/lox) mouse embryonic fibroblast cells from Ecd floxed mouse embryos. Cre-mediated deletion of Ecd in Ecd(lox/lox) mouse embryonic fibroblasts led to a proliferative block due to a delay in G(1)-S cell cycle progression; this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Furthermore, Ecd directly bound to Rb …

Disruption Of C-Jun Reduces Cellular Migration And Invasion Through Inhibition Of C-Src And Hyperactivation Of Rock Ii Kinase., Xuanmao Jiao, Sanjay Katiyar, Manran Liu, Susette C Mueller, Michael P. Lisanti, Anping Li, Timothy G Pestell, Kongming Wu, Xiaoming Ju, Zhiping Li, Erwin F Wagner, Tatsuo Takeya, Chenguang Wang, Richard G Pestell

Kimmel Cancer Center Faculty Papers

The spread of metastatic tumors to different organs is associated with poor prognosis. The metastatic process requires migration and cellular invasion. The protooncogene c-jun encodes the founding member of the activator protein-1 family and is required for cellular proliferation and DNA synthesis in response to oncogenic signals and plays an essential role in chemical carcinogenesis. The role of c-Jun in cellular invasion remains to be defined. Genetic deletion of c-Jun in transgenic mice is embryonic lethal; therefore, transgenic mice encoding a c-Jun gene flanked by LoxP sites (c-jun(f/f)) were used. c-jun gene deletion reduced c-Src expression, hyperactivated ROCK II signaling, …

Gene Targeting In Adult Rhesus Macaque Fibroblasts, Daniel T. Meehan, Mary Ann Zink, Melissa Mahlen, Marilu Nelson, Warren G. Sanger, Shoukhrat M. Mitalipov, Don P. Wolf, Michel M. Ouellette, Robert B. Norgren

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT) has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology.

RESULTS: A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow …

Cpg Hypomethylation In A Large Domain Encompassing The Embryonic Β-Like Globin Genes In Primitive Erythrocytes, Mei Hsu, Rodwell R. Mabaera, Christopher H. Lowrey, David I. K. Martin, Steven Fiering

Dartmouth Scholarship

There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the β-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine β-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large (∼20-kb) …

Smc3 Knockdown Triggers Genomic Instability And P53-Dependent Apoptosis In Human And Zebrafish Cells., Giancarlo Ghiselli

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: The structural maintenance of chromosome 3 (SMC3) protein is a constituent of a number of nuclear multimeric protein complexes that are involved in DNA recombination and repair in addition to chromosomal segregation. Overexpression of SMC3 activates a tumorigenic cascade through which mammalian cells acquire a transformed phenotype. This has led us to examine in depth how SMC3 level affects cell growth and genomic stability. In this paper the effect of SMC3 knockdown has been investigated. RESULTS: Mammalian cells that are SMC3 deficient fail to expand in a clonal population. In order to shed light on the underlying mechanism, experiments …

Selective Role For Superoxide In Insp3 Receptor-Mediated Mitochondrial Dysfunction And Endothelial Apoptosis., Muniswamy Madesh, Brian J Hawkins, Tatyana Milovanova, Cunnigaiper D Bhanumathy, Suresh K Joseph, Satish P Ramachandrarao, Kumar Sharma, Tomohiro Kurosaki, Aron B Fisher

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane …

Functionally Active T1-T1 Interfaces Revealed By The Accessibility Of Intracellular Thiolate Groups In Kv4 Channels., Guangyu Wang, Mohammad Shahidullah, Carmen A Rocha, Candace Strang, Paul J Pfaffinger, Manuel Covarrubias

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is …

Erythroid Cell-Specific Α-Globin Gene Regulation By The Cp2 Transcription Factor Family, Ho C. Kang, Jui Hyung Chae, Yeon H. Lee, Mi-Ae Park, June Ho Shin, Sung-Hyun Kim, Sang-Kyu Ye, Yoon Shin Cho, Steven Fiering, Chul Geun Kim

Dartmouth Scholarship

We previously demonstrated that ubiquitously expressed CP2c exerts potent erythroid-specific transactivation of alpha-globin through an unknown mechanism. This mechanism is reported here to involve specific CP2 splice variants and protein inhibitor of activated STAT1 (PIAS1). We identify a novel murine splice isoform of CP2, CP2b, which is identical to CP2a except that it has an additional 36 amino acids encoded by an extra exon. CP2b has an erythroid cell-specific transcriptional activation domain, which requires the extra exon and can form heteromeric complexes with other CP2 isoforms, but lacks the DNA binding activity found in CP2a and CP2c. Transcriptional activation of …

Fibroblast Growth Factor 2 Endocytosis In Endothelial Cells Proceed Via Syndecan-4-Dependent Activation Of Rac1 And A Cdc42-Dependent Macropinocytic Pathway, Eugene Tkachenko, Esther Lutgens, Radu-Virgil Stan, Michael Simons

Dartmouth Scholarship

Full activity of fibroblast growth factors (FGFs) requires their internalization in addition to the interaction with cell surface receptors. Recent studies have suggested that the transmembrane proteoglycan syndecan-4 functions as a FGF2 receptor. In this study we investigated the molecular basis of syndecan endocytosis and its role in FGF2 internalization in endothelial cells. We found that syndecan-4 uptake, induced either by treatment with FGF2 or by antibody clustering, requires the integrity of plasma membrane lipid rafts for its initiation, occurs in a non-clathrin-, non-dynamin-dependent manner and involves Rac1, which is activated by syndecan-4 clustering. FGF2 was internalized in a complex …

Fibroblast Growth Factor–Specific Modulation Of Cellular Response By Syndecan-4, Arie Horowitz, Eugene Tkachenko, Michael Simons

Dartmouth Scholarship

Proteoglycans participate in growth factor interaction with the cell surface through their heparan sulfate chains (HS), but it is not known if they are otherwise involved in growth factor signaling. It appears now that the syndecan-4 core protein, a transmembrane proteoglycan shown previously to bind phosphatidylinositol 4,5-bisphosphate (PIP(2)) and activate PKC alpha, participates in mediating the effects of fibroblast growth factor (FGF)2 on cell function. Mutations in the cytoplasmic tail of syndecan-4 that either reduced its affinity to PIP(2) (PIP(2)(-)) or disrupted its postsynaptic density 95, disk large, zona occludens-1 (PDZ)-dependent binding (PDZ(-)) produced a FGF2-specific dominant negative phenotype in …

Distinct Retrieval And Retention Mechanisms Are Required For The Quality Control Of Endoplasmic Reticulum Protein Folding, Shilpa Vashist, Woong Kim, William J. Belden, Eric D. Spear, Charles Barlowe, Davis T.W. Ng

Dartmouth Scholarship

Proteins destined for the secretory pathway must first fold and assemble in the lumen of endoplasmic reticulum (ER). The pathway maintains a quality control mechanism to assure that aberrantly processed proteins are not delivered to their sites of function. As part of this mechanism, misfolded proteins are returned to the cytosol via the ER protein translocation pore where they are ubiquitinated and degraded by the 26S proteasome. Previously, little was known regarding the recognition and targeting of proteins before degradation. By tracking the fate of several mutant proteins subject to quality control, we demonstrate the existence of two distinct sorting …

Interleukin-2-Triggered Raf-1 Expression, Phosphorylation, And Associated Kinase Activity Increase Through G1 And S In Cd3-Stimulated Primary Human T Cells., Antanina Zmuidzinas, Harvey J. Mamon, Thomas M. Roberts, Kendall A. Smith

Dartmouth Scholarship

To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion …