The Pennsylvania State University The Graduate School College of Medicine A ROLE FOR STANNIN IN CELLULAR SIGNALING A Thesis in Integrative Biosciences by Brian Reese 2005 Brian Reese Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2005 The thesis of Brian Eric Reese was reviewed and approved* by the following: Melvin L. Billingsley Professor of Pharmacology Thesis Advisor Co-Chair of Committee Jong K. Yun Assistant Professor of Pharmacology Co-Chair of Committee Robert J. Milner Professor of Neural and Behavioral Sciences James R. Connor Professor and Vice-Chair of Neurosurgery J. Kyle Krady Assistant Professor of Neural and Behavioral Sciences Anita K. Hopper Professor of Biochemistry and Molecular Biology Co-Director of Integrative Biosciences Graduate Program *Signatures are on file in the Graduate School ABSTRACT Trimethyltin (TMT) is a selective and potent neurotoxicant capable of inducing apoptotic cell death in the hippocampal formation, neocortex, amygdala, and olfactory tubercle. Subtractive hybridization was used in previous studies to uncover any common gene products present in TMT-sensitive tissues that might account for TMT’s selective toxicity. These studies showed that the gene product Stannin (Snn) was preferentially expressed in TMT sensitive tissues, with higher levels of Snn expression correlating with higher regional levels of TMT sensitivity. In addition, using antisense oligonucleotides, Snn was found to be necessary, but not sufficient, for TMT toxicity. Snn is an 88 amino acid, membrane-bound protein with a molecular weight of 9.497 kDa. Snn is found in vertebrate species and is highly conserved across vertebrates, with human and rat Snn showing 98% identity at the amino acid level. Snn is widely expressed in the developing embryo; Snn expression becomes more restricted during maturation to adulthood. In the adult, Snn is expressed in the spleen, immune cells, brain, kidney and lung. Snn has no significant homology to any other known protein. Tumor necrosis factor alpha (TNFα) was shown to induce Snn mRNA expression in human umbilical vein endothelial cells (HUVECs). We used quantitative, real-time PCR (QRT-PCR) to quantify the induction of Snn by TNFα in multiple cell lines. We observed significant increases in Snn mRNA in a time-dependent manner in HUVECs and Jurkat T-cells. To better define a potential mechanism underlying this induction, chemical inhibitors of protein kinase C (PKC) were used to determine if PKC played a role in TNFα-mediated Snn gene expression. The results of these experiments indicated iii that one or more of the βII, δ, γ, or ε isoforms was responsible for TNFα-mediated Snn gene expression. To determine specifically which isoform(s) of PKC were involved, we utilized short interfering RNA technology (siRNA) and found that PKCε was responsible for mediating TNFα-induced Snn gene expression. TMT can induce the expression of TNFα in mixed neuronal/glial cultures and so we hypothesized that TMT exposure would cause an increase in Snn gene expression, potentially as a necessary component of TMT toxicity. Again, QRT-PCR was used and we found that TMT significantly upregulated Snn within 9 hours of exposure. However, blocking TNFα with neutralizing antibodies resulted in only partial protection against TMT. By placing Snn in a defined TNFα-PKCε signaling pathway, several hypotheses arise concerning the function of Snn, such as Snn being part of a cell death or cell survival-signaling pathway. Given the high level of conservation of Snn, generation of high-affinity, Snn-specific antisera has proven difficult. In order to assess a potential role for Snn in the HUVEC response to TNFα, microarray technology was used. We found that knocking Snn down via siRNA significantly altered HUVEC gene expression in response to TNFα. After normalization and statistical analysis, we found that several genes altered by Snn knockdown are involved in the modulation of the cell cycle and cell growth. Specifically, several genes are involved with p53 and Cyclin D1, known G1/S checkpoint proteins. Functional assays showed that Snn knockdown resulted in significantly less HUVEC growth relative to other treatment. Further, analysis via flow iv cytometry indicated that a significant portion of TNFα-, Snn siRNA co-treated HUVECs were halted in the G1 phase of the cell cycle. Together, the data presented outline a signaling pathway leading to enhanced Snn gene expression as well as a potential role for Snn in normal cellular function. A role in modulating the cell cycle would explain the high degree of conservation of Snn across vertebrate evolution as well as the tissue-specific pattern of expression of Snn during different life stages. This work details the first known interaction of Snn in a cellular signaling pathway as well as the first evidence of a potential functional role of Snn in normal cells. v TABLE OF CONTENTS List of Figures..............................................................................................................…ix List of Tables ...............................................................................................................…xi List of Abbreviations …………………………………………………………………..xii Acknowledgements.........................................................................................................xiv Chapter 1: Introduction……………………………………………………………………1 Chapter 2: Literature Review……………………………………………………………..4 A. Trimethyltin 1. Organotins as toxicants…………………………………………………4 2. Trimethyltin - a potent, selective neurotoxicant………………………..5 3. Trimethyltin toxicity - cellular mechanisms……………………………6 4. Trimethyltin toxicity - behavioral effects………………………………8 5. Trimethyltin - summary…………………………………………….…..8 B. Stannin 1. The discovery of stannin and characterization of expression…………..9 2. Characterization of the stannin gene…………………………………..10 3. Stannin's role in trimethyltin toxicity……………………………….…13 4. Stannin in cellular signaling…………………………………………...14 C. Tumor necrosis factor-α 1. Tumor necrosis factor-α - origin and plieotropism…………...………17 2. Tumor necrosis factor-α receptor 1: signal transduction and major functions………………………………………………………..17 3. Tumor necrosis factor-α receptor 2: signal transduction and known functions…………………………………………………….…21 D. Protein Kinase C 1. Classes of protein kinase c and activation………………………….…22 2. Functions of protein kinase c…………………………………….……25 3. Protein kinase C - summary………………………………………..….30 Chapter 3: Protein Kinase C Epsilon Regulates TNFα-Induced Stannin Gene Expression……………………………………………………………………31 A. Introduction……………………………………………………………...……31 B. Methods 1. Cell culture………………………………………………………...…..35 2. Reagents…………………………………………………………...…..35 3. Cell viability…………………………………………………………...36 vi 4. RNA isolation/cDNA synthesis………………………………………36 5. Quantitative real-time PCR……………………………………...……37 6. siRNA construction………………………………………………...…37 7. Transfection of siRNA…………………………………………..……39 8. Statistical analysis………………………………………………..…...39 C. Results……………………………………………………………………..…40 1. Tumor necrosis factor-α contributes to trimethyltin toxicity…….…..40 2. Trimethyltin induces stannin gene expression in human umbilical vein endothelial cells……………………………………....40 3. Stannin knockdown rescues HUVECs from trimethyltin toxicity..….41 4. Induction of stannin mRNA by tumor necrosis factor-α………….…47 5. Protein kinase c is required for TNFα-induced stannin mRNA expression…………………………………………………....52 6. Knockdown of protein kinase c epsilon via siRNA prevents TNFα-mediated stannin upregulation………………………………..55 D. Discussion………………………………………………………………..…..59 Chapter 4: Microarray Analysis of Stannin Knockdown in Human Umbilical Vein Endothelial Cells in TNFα Response; Implications for Cell Cycle Control…………………………………………………………………….....65 A. Introduction………………………………………………………………......65 B. Methods 1. Cell culture……………………………………………………………67 2. Microarray fabrication………………………………………………..67 3. Microarray cDNA probe synthesis and indirect labeling with AlexaFluor555 and 647…………………………………………67 4. Gene expression analysis……………………………………………..68 5. Cell Growth…………………………………………………………...69 6. RNA isolation/cDNA synthesis………………………………………69 7. Quantitative real-time PCR…………………………………………...70 8. siRNA construction…………………………………………………...70 9. Transfection of siRNA………………………………………………..71 10. Statistical analysis……………………………………………..….…72 C. Results 1. Stannin knockdown results in significantly altered HUVEC gene expression in response to TNFα……………………………..…73 2. Stannin knockdown significantly alters several genes involved in cell growth……………………………………………………..…..76 vii 3. Loss of stannin gene expression functionally affects HUVEC response to TNFα………………………………………………..…84 4. Knockdown of stannin inhibits the ability of TNFα-treated HUVECs to progress through the cell cycle…………………….….87 D. Discussion……………………………………………………………….…92 Chapter 5: Discussion…………………………………………………………………95 A. Stannin expression is highly regulated in a spatial and temporal manner…95 B. Stannin as a mediator of trimethyltin toxicity……………………………...96 C. Stannin as a component of cell cycle progression…………………………98 D. Overall Conclusions……………………………………………………….106 References……………………………………………………………………………..102 Appendix - Complete list of significantly altered genes from the microarray………..118 viii LIST OF FIGURES 1. Developmental alteration in stannin expression pattern……………………………..12 2. Northern blotting analysis for the expression of 5 novel cytokine-responsive genes in resting and TNFα-activated