A Role for Stannin in Cellular Signaling
Open Access
- Author:
- Reese, Brian
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 18, 2005
- Committee Members:
- Melvin Lee Billingsley, Committee Chair/Co-Chair
Jong Kak Yun, Committee Chair/Co-Chair
Robert J Milner, Committee Member
James Robert Connor, Committee Member
J Kyle Krady, Committee Member - Keywords:
- stannin
TNFalpha
PKC
Trimethyltin - 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 (TNFa) 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 TNFa 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 TNFa-mediated Snn gene expression. The results of these experiments indicated that one or more of the bII, d, g, or e isoforms was responsible for TNFa-mediated Snn gene expression. To determine specifically which isoform(s) of PKC were involved, we utilized short interfering RNA technology (siRNA) and found that PKCe was responsible for mediating TNFa-induced Snn gene expression. TMT can induce the expression of TNFa 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 TNFa with neutralizing antibodies resulted in only partial protection against TMT. By placing Snn in a defined TNFa-PKCe 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 TNFa, microarray technology was used. We found that knocking Snn down via siRNA significantly altered HUVEC gene expression in response to TNFa. 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 cytometry indicated that a significant portion of TNFa-, 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.