Signal Transduction in Blood Cells, Normal and Leukemic

Lab memebers (L-R): Stacy McMahon, George Auzani, Julian G. Cambronero, Mauricio Di Fulvio and Nick Lehman. (Other lab members pictured below are (L-R): Jennifer Hollyfield, Isabel Campos, and Nick McCray.) Research interests: Molecular cell signaling in human neutrophils as elicited by hematopoietic growth factors; cell migration (chemotaxis); pathological connections with inflammation-mediated tissue injury and with leukemia; new methodologies for the study of protein-protein interaction and enzyme inhibitor design. Research lines: Neutrophil/Endothelium/Pathogen injury & atherogenesis 2: Neutrophil PLD, oxygen radicals, & ischemia/reperfusion 3: Signal transduction pathways in leukemic cells

Principal investigator:   Julian Gomez-Cambronero, Ph.D. Professor, Cell Biology and Physiology Director, Blood/Hematology Course Boonshoft School of Medicine Wright State University Phone: (937) 775-2360 Fax: (937) 775-3391 julian.cambronero@wright.edu Laboratory members:   Mauricio Di Fulvio, Ph.D. (postdoctoral fellow) Jennifer Hollyfield (Ph.D. student) Stacy McMahon (M.S. student) Nick McCray (research assistant) Nick Lehman (biology major) Isabel Campos (biology major) George Auzani (medical student) Collaborators at WSU:   Michael A. Baumann, M.D. (Medicine) Cassandra C. Paul, Ph.D. (Medicine) Nancy J. Bigley, Ph.D. (Immunology) Mill W. Miller, Ph.D. (Biology) Ken Turnbull, Ph.D. (Organic Chemistry) Collaborators at other universities:   J. Joseph Blum, Ph.D. (Duke University) Linda McPhail, Ph.D. (Wake Forest University School of Medicine) Mary C. Dinauer (Indiana University School of Medicine) Vital Signsarticle:"Researchers Seek to Unravel a Central Paradigm in Cell Biology,"Fall 2004. News release: "Tribute Given to Dr. Gomez-Cambronero,"March 2005 News release:"WSU Researcher at the Forefront of a New Field,"March 2004

Research line 1:
Neutrophil/Endothelium/Pathogen injury and atherogenesis

For some time, researchers have been awestruck with the fact that nearly 50 percent of the people having atherosclerosis and heart attacks had normal levels of cholesterol and oxidized LDL and followed healthy lifestyles. In the last few years, investigators seem to have identified a new "culprit" for the remaining 50 percent: Inflammation. The connection of the immune system's powerful inflammatory response with cardiopathies are now being increasingly recognized. Further, there is considerable evidence that pathogens from the oral flora can cause damage in coronary arteries (the Red Cross will not accept a blood donation from anyone who has had a dental cleaning in the previous 24 hours).

Based on this, we have proposed that bacterial infectionof endothelial cells lining the coronary artery's wall will create a focus of infection. This will attract neutrophils from the bloodstream. The neutrophils' mission is to seek and destroy invading pathogens. Initially, neutrophils will attempt to eliminate the invading pathogen in the artery by the release of powerful proteases and reactive oxygen species. But in doing so, these very substances will inflict collateral damage on the endothelial and fibroblast walls. The problem is that in predisposed individuals, this process might become chronic, particularly with the recruitment and the arrival of more leukocytes and the overall amplification by numerous inflammatory signals (cytokines). If the process does not resolve appropriately, this "battlefield" will leave a "scar" of damaged tissue in the lining of the artery which will serve as the nidus for a nascent atherosclerotic plaque.

Pathogens, inflammation and atherogenesis hypothesis

We have proposed to develop and establish a "Neutrophil-Endothelial Cell-Pathogen" (N EC-P) model. This represents a completelynovel experimental model of an artery wall for the purpose of identifying chemoattractant molecules released by infected EC and assessing the enzymatic damage produced by activated neutrophils. This will comprise:

[a]Bacterially-infected endothelial cells (ECs) with S. sanguisand S. epidermidis, [b]ECs' release of cytokines IL-8 and GM-CSF, [c]Neutrophil infiltration to the site of infection, [d]Destruction of bacteria by myeloperoxidase (MPO) and other lytic enzymes, and [e] Collateral damage inflicted upon ECs and other cells (namely, fibroblasts and smooth muscle cells).

This model will provide new molecular targets for the interaction of infected vascular endothelium and cells of the innate immune system. These in turn could be used to design treatments to prevent neutrophil mediated tissue injury that occurs in atherosclerosis.

Techniques for this research

In vitromodels of atherogenesis; cell co-cultures; cDNA array techniques to pinpoint the genes involved in cytoskeletal-related signaling pathways; co-immunoprecipiation and in vitrokinase assays; forced expression of genes and their effect on parallel signaling pathways; transfection of dominant negative cDNA, antisense oligonucleotides and renatured proteins and assessment of pathophysiological effects.

Research line 2:
Neutrophil phospholipase D (PLD), oxygen radicals, and ischemia/reperfusion injury

We are seeking to understand the molecular mechanism underlying the production of lethal oxygen radicals duringischemia and reperfusion injury. Our studies focus on the enzyme phospholipase D (PLD) that is central to the production of phosphatidic acid (PA), a putative second messenger involved in the release of superoxide anions by neutrophils' respiratory burst (NADPH oxidase system). There are two fronts of attack in this line:

[a] To purify and clone neutrophil PLD, which we propose to be a distinctive form from the described mammalian PLD1 and PLD2 isoforms.Our first aim is to purify the enzyme by fast protein liquid chromatography (FPLC) and by molecular biology techniques, using the N-terminal 15-20 amino acids of the sequenced purified protein as well as the existing mammalian PLD cDNA sequences (PLD1 and PLD2). Our second aim is to determine the mechanism of granulocyte PLD regulation, particularly the phosphorylating kinase, the site(s) of phosphorylation and the role of small GTP-binding proteins, using immunoprecipitation with antibodies against epitope-tagged proteins and in vitroenzymatic assays.

Our working hypothesis is that a novel isoform of PLDin human neutrophils is regulated through tyrosine phosphorylation, which allows the enzyme to become active and synthesize phosphatidic acid (PA), that in turn triggers the release of toxic oxygen radicals. These are responsible for the exacerbation of tissue damage and necrosis after a heart attack.

[b] To find a selective inhibitor of PLD,intended to ameliorate the devastating effectsof the ischemia/reperfusion pathological condition. This will be done in the following sequential steps:

(1) Starting from the serine protease inhibitor AEBSF, and using known organic synthesis methods, we will synthesize a phospholipase D (PLD) inhibitor able to enter a living cell through its membrane;

(2) We will determine the particular site in the PLD molecule to which the inhibitor binds, and the mechanism of inhibition. We will do this by protein chemistry and molecular biology methods;

(3) We will use the newly synthesized enzyme inhibitorto asses its effect in vivo(intact cells) and quantify the contribution of PLD to reperfusion injury. An inhibitor of this class will potentially block the lethal effect of free oxygen radicals that cause neutrophil-derived damage to heart tissue.

Techniques for this research

Organic synthesis by combinatorial chemistry methods; fluorography; amino acid and tryptic peptide analyses; peptide arrays; generation of protein single-point mutants; three-dimensional structure modeling and other proteomics methodologies on protein-protein interaction and enzyme inhibitor design.

Funding

This research line is funded by the American Heart Association (National Program).

Research line 3:
Signal transduction pathways in leukemic cells

We are seeking to understand the signal transduction mechanisms that are initiated by the binding of the granulocyte-macrophage colony stimulating factor (GM-CSF) to human leukocytes (leukemic blasts and mature neutrophils).

This hematopoietic growth factor is currently used to restore the normal white blood cells circulating levels in patients undergoing chemotherapyand during bone marrow transplantation. GM-CSF stimulates major signaling pathways such as mitogen-activated protein kinases (MAPK) p42ERK2 and p44ERK1, which become dually-phosphorylated on tyrosine and serine/threonine; and the ribosomal S6 kinases p90rsk and p70S6K.

Positive feedback of MAPK on p70s6k pathway leading to heightened chemotaxis

Using proleukemic cell lines of the myelocytic lineage as the biological source, we are currently studying:

[a]the point at which MAP and S6 kinases are turned on/off during the late stages of white blood cell maturation; and

[b]the physiological role of MAPK upregulation in mature cells. To this, our data aim at a molecular cross-talkbetween MAPK and p70S6K signaling pathways.

We propose that upon commitment to hematopoietic differentiation, the normal transduction pathways switch from cell-dividing mechanisms to a way that allows the cell to mount a full response (e.g., chemotaxis and phagocytosis) against invading pathogen during infection.

Techniques for this research

Leukemic cell culture; immunochemistry (phosphotyrosine analyses); RT-PCR; cytopreprarations; flow cytometry; immunofluorescence microscopy; enzyme assays (radiometric, photometric and in-gel); phagocyte functional assays.

Funding

This research line is funded by Wright State University Boonshoft School of Medicine and the National Institutes of Health (NIH-NHLBI).

Dr. Julian Gomez-Cambronero

Principal investigator's biography

Dr. Julian Gomez-Cambronero received his Ph.D. (cum laude) in Biochemistry/Immunology at the Complutense University (Madrid, Spain) in 1986 under Drs. M. Sanchez Crespo and J. M. Mato's direction. He then moved to the United States and completed his training in Cell Biology as a postdoctoral fellow in Dr. R. I. Sha'afi's laboratory at the Department of Physiology of the University of Connecticut Health Center, and with the late Dr. E. L. Becker. From 1991 to 1995 he was part of the faculty at the University of Connecticut, first as an Instructor and then as a Research Assistant Professor. In 1995 he moved to the Department of Physiology and Biophysics at Wright State University, chaired by Dr. P.K. Lauf, to accept a tenure-track Assistant Professorship position, and was promoted to Associate Professor in 2000 and to full Professor in 2004. He is married to Teresa Madrid and has two children, David and Julia.

Member of the graduate program

As part of the graduate faculty and as a dissertation-qualified professor, he participates in the following areas of concentration of the Biomedical Sciences (BMS) Doctoral (Ph.D.) Program: Cell Biology and Physiology, Immunology, and Biochemistry and Molecular Biology. He also participates in the Master of Science (MS) Program in Physiology and Biophysicsand the Immunology MS Program. His formal lectures include an "Introduction to Hematology" for first year medical students in the team-taught Molecular, Cellular and Tissue Biology course; "Molecular Basis of Leukemia and Lymphoma" second year medical students; and "Signal Transduction/Molecular Endocrinology and Blood Physiology" for the BMS Ph.D. Program. He is course director for "Blood/Hematology" for the School of Medicine.

Honors and awards

Dr. G-Cambronero is a full member of the American Physiological Society, the American Society for Biochemistry and Molecular Biology, the American Society of Hematology, and the Society for Leukocyte Biology. He received an American Heart Association (AHA) Postdoctoral Fellowship in 1987; was part of the Leukemia Society of America (LSA) Northern Connecticut Chapter Telecast in 1990; was National Finalist for the Young Investigator Award of the Society for Leukocyte Biology (SLB) in 1992; received the New Investigator Research Award from the Donaghue Medical Research Foundation in 1992; was elected as Expert Scientist by the Farmington, Connecticut, Public School Board to evaluate the science curriculum in 1994; was the subject of a biographical record in the American Men & Women of Science directory in 1995; received the Frontiers in Physiology National Research Award from the American Physiological Society (APS) in 1996; was recognized with the STARS Scholar Distinguished Service Award by The Ohio University in 1998; received the Professional Achievement "Sembrador" Award from The City of Manzanares (CR), Spain, in 2004; and received the Outstanding Achievement in Medical Education and Research Award from the Academy of Medicine of Dayton in 2007.

Funding and scholarly publications

Dr. G-Cambronero has received funding from the National Institutes of Health (NIH NHLBI); the American Cancer Society (ACS); the American Heart Association (National Program); the American Physiological Society; the Ohio Board of Regents; the University of Connecticut; and Wright State University.

He has been invited to the Erythrocyte and Leukocyte Biology (ELB), Cell Development and Function (CDF-2), and Innate Immunity and Inflammation (III) Study Sections for the NIH; and to the Molecular Signaling, Basic Cell and Molecular Biology Review Panel for the American Heart Association
(AHA) Research Consortium.

He has published over 60 original research papers in peer-review journals, five book chapters, and has presented over 50 research posters and seminar talks both nationally and internationally. He has also published science-fiction short stories and layman science-related articles in Spanish literary magazines.

PUBLICATIONS

Representative of Doctorate Period:

Gomez-Cambronero, J., Mato, J.M., Vivanco, F. and Sanchez-Crespo, M. (1987) Phosphorylation of partially purified 1-0-alkyl-2-lyso-glycero-3-phosphocholine: acetyl-CoA acetyltransferase from rat spleen. Biochem. J.245, 893-898. PDF

Representative of Postdoctoral Period:

Gomez-Cambronero, J., Huang, C-K., Gomez-Cambronero, T.M., Waterman, W.H., Becker, E.L. and Sha'afi, R.I. (1992) Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of MAP Kinase in human neutrophils. Proc. Natl. Acad. Sci.USA. 89, 7551-7555. PDF

As Principal Investigator:

Johnson, G.M. and Gomez-Cambronero, J. (1995) Priming of tyrosine phosphorylation in GM-CSF-stimulated adherent neutrophils. J. Leukocyte Biol.57, 692-698. Abstract

Gomez-Cambronero, J. (1995) Immunoprecipitation of a phospholipase D activity with anti-phosphotyrosine antibodies. J. Interferon Cytok. Res.15, 877-885. Abstract

Joseph, D., Paul, C.C., Baumann, M.A. and Gomez-Cambronero, J. (1996) S6 kinase p90rsk in GM-CSF-stimulated proliferative & mature hemopoietic cells. J. Biol. Chem.271, 13088-93. PDF

Gomez-Cambronero, J. and Veatch, C. (1996) Emerging paradigms in granulocyte macrophage colony-stimulating factor signaling. Life Sci.59, 2099-2111. Abstract

Gomez-Cambronero, J. and Keire, P. (1998) Phospholipase D: a novel major player in signal transduction. Cell Signal. 10, 387-397. Abstract

Hayes, T.S., Billington, C.J., Robinson, K.A., Sampt, E.R., Fernandez, G.A. and Gomez Cambronero, J. (1999) Binding of granulocyte-macrophage colony-stimulating factor to adherent neutrophils activates Phospholipase D. Cell Signal.11, 195-204. Abstract

Gomez-Cambronero, J. (1999) MAP kinase is activated in EGF-stimulated interphase but not in mitotic HeLa cells. FEBS Lett.443, 126-130. PDF

Grishin, A., Sinha, S., Roginskaya, V., Boyer, M.J., Gomez-Cambronero, J., Zuo, S., Kurosaki, T., Romero, G. and Corey S.J. (2000) Involvement of Shc and Cbl-PI 3 kinase in Lyn-dependent proliferative signaling pathways for G-CSF. Oncogene19, 97 105. Abstract

Andrews, B., Bond, K., Lehman, J.A., Horn, J.M., Dugan, A. and Gomez-Cambronero, J. (2000) Direct inhibition of in vitro PLD activity by 4-(2-aminoethyl) benzenesulfonyl fluoride. Biochem. Biophys. Res. Com.273, 302-311. Abstract

Paul, C.C., Aly, E., Lehman, J.A., Page, S.M., Gomez-Cambronero, J., Ackerman, S.J. and Baumann, M.A. (2000). Human cell line that differentiates to all myeloid lineages and expresses neutrophil secondary granule genes. Exp. Hematol.28, 1373-1380. Abstract

Lehman, J.A., Paul, C.C., Baumann, M.A. and Gomez-Cambronero, J. (2001) MAP kinase upregulation after hematopoietic differentiation: role of chemotaxis. Am. J. Physiol. Cell Physiol.280, 183-191. PDF

Horn, J.M., Lehman, J.A., Alter, G., Horwitz, J. and Gomez-Cambronero, J. (2001) Presence of a phospholipase D (PLD) distinct from PLD1 or PLD2 in human neutrophils: immuno-biochemical characterization and initial purification. Biochim. Biophys. Acta1530, 97-110. Abstract

Blum, J.J., Lehman, J.A., Horn, J.M. and Gomez-Cambronero, J. (2001) Phospholipase D (PLD) is present in L. donovaniand its activity increases in response to acute osmotic stress. J. Eukaryotic Microbiol.48, 102-110. Abstract

Sampt, E.R., Fernandez, G.A., Lehman, J.A., Corey, S.J., Huang, C.-K. and Gomez Cambronero, J. (2001) A systematic approach to the complete study of a signaling molecule: ribosomal p90rsk as an example.J. Biochem. Biophys. Meth.48, 219-237. Abstract

Gomez-Cambronero, J. (2001) The oxygen dissociation curve of hemoglobin: bridging the gap between Biochemistry and Physiology. J. Chem. Ed.78, 757-759. Abstract

Baumann, M.A., Paul, C.C., Lemley-Gillespie, S., Oyster, M. and Gomez-Cambronero, J. (2001) Modulation of MEK activity during G-CSF signaling alters proliferative vs.differentaitive balancing.Am. J. Hematol.68, 99-105. Abstract

Lehman, J.A. and Gomez-Cambronero, J. (2002) Molecular crosstalk between p70S6K and MAPK cell signaling pathways. Biochem. Biophys. Res. Com.293, 463-469. Abstract

Gomez-Cambronero, J., Horwitz, J. and Sha'afi, R.I. (2003) Measurements of Phospholipases A2, C and D (PLA2, PLC and PLD): in vitro microassays, analysis of enzyme isoforms and intact cells assays. Methods Mol. Biol.218, 155-1576. Abstract

Lehman, J.A., Calvo, V. and Gomez-Cambronero, J. (2003) Mechanism of ribosomal p70S6 kinase activation by GM-CSF in neutrophils: Cooperation of a MEK-related, T421/S424-kinase and a rapamycin-sensitive, mTOR-related, T389-kinase. J. Biol. Chem.278, 28130-28138. PDF

Gomez-Cambronero, J. (2003) Rapamycin inhibits GM-CSF-induced neutrophil migration. FEBS Let.550, 94-100. Abstract

Gomez-Cambronero, J., Horn, J., Paul, C.C. and Baumann, M.A. (2003) GM-CSF is a chemoattractant cytokine for neutrophils: Involvement of the ribosomal p70S6K signaling pathway. J. Immunol.171, 6846-6855. Abstract

Gomez-Cambronero, J., Frye, T. and Baumann, M.A. (2004) Ribosomal p70S6K basal activity increases upon induction of differentiation of myelomonocytic leukemic cell lines HL60, AML14 and MPD. Leukemia Res.28, 755-62. Abstract

Baumann M, Frye T, Naqvi T and Gomez-Cambronero J. (2005) Normal neutrophil maturation is associated with selective loss of MAP kinase activation by G-CSF. Leukemia Res.29, 73-8. Abstract

Di Fulvio, M. and Gomez-Cambronero, J. (2005) Phospholipase D gene expression in human neutrophils and HL-60 differentiation. J. Leukocyte Biol.77, 999-1007. Abstract

Horn, J., Miller, M. Lopez, I. and Gomez-Cambronero, J. (2005) The uncovering of a novel regulatory mechanism for PLD2: Formation of a ternary complex with Protein Tyrosine Phosphatase PTP1b and Growth Factor Receptor-Bound Protein GRB2. Biochem. Biophys. Res. Com.332, 58-67. Abstract

Di Fulvio, M., Lehman, N., Xiaohong Lin, X, Lopez, I. and Gomez-Cambronero, J. (2006) The elucidation of novel SH2 binding sites on PLD2. Oncogene 25, 3032-3040. Abstract.

Lehman N, Di Fulvio M, McCray N, Campos I, Tabatabaian F, Gomez-Cambronero J. (2006) Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2. Blood108, 3564-72. Abstract.

Lehman N, Ledford B, Di Fulvio M, Frondorf K, McPhail LC, Gomez-Cambronero J. (2007) Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR. FASEB J 21, 1075-1087. Abstract.

Di Fulvio M, Frondorf K, Henkels KM, Lehman N, Gomez-Cambronero J. (2007) The Grb2/PLD2 interaction is essential for lipase activity, intracellular localization and signaling in response to EGF. J Mol Biol. 367, 814-824. Abstract.

Gomez-Cambronero J, Di Fulvio M, Knapek K. (2007) Understanding phospholipase D (PLD) using leukocytes: PLD involvement in cell adhesion and chemotaxis. J Leukoc Biol. 82, 272- 281. Abstract.

Di Fulvio M, Henkels KM, Gomez-Cambronero J. (2007) Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis. Biochem Biophys Res Commun. 357, 737-742. Abstract.

Fulvio MD, Frondorf K, Gomez-Cambronero J. (2008) Mutation of Y(179) on phospholipase D2 (PLD2) upregulates DNA synthesis in a PI3K-and Akt-dependent manner. Cell Signal. 20, 176-185. Abstract.