ST 18 regulates TNF-alpha induced apoptosis and inflammatory gene expression
Yang, Yi Julia
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TNF-alpha regulates inflammation the expression of other genes through activation of transcript factots such as NF-kB, AP-1 and CREB. TNF-alpha is a one of the prime signals that induces apoptosis in various types of cells. TNF-alpha induced apoptosis is one of the principle mechanisms by which cells are removed. TNF-alpha is also a potent inflammatory mediator, which in turn activates a number of pro-inflammatory molecules through transcriptional regulation. A prototype of such a transcription factor regulating both inflammation and apoptosis is NF-kB. However it has anti-apoptotic effect and cannot account for the pro-apoptotic effect of TNF-alpha. Therefore other transcription factors are likely to be involved in this process, formally known as NZF-3, KIAA0535 or ZNF387) is located on 8 q 11 region of chromosome 8. It was first isolated and identified in the rat brain using degenerate primers. However, its function as a transcription factor regarding its target genes has not been investigated. Our previous protein/DNA array results showed that protein level of ST18 was elevated 4 fold upon stimulation of TNF-alpha. The purpose of this study is to investigate the role of ST 18 in mediating TNF-alpha-induced apoptosis and inflammatory gene expression. Adult dermal fibroblasts were stimulated in vitro with 20ng/ml of the recombinant human TNF-alpha for 0, 1 and 3 hours. Nuclear proteins were extracted and EMS A (Electrophoretic Mobility Shift Assay) was performed to evaluate promoter binding interactions (protein/DNA probe). EMSA results showed that ST 18 nuclear activation was elevated 1 hour after TNF-a stimulation, indicating protein/DNA interaction. The level of activation returned to baseline after 3 hours. Our real-time PCR result also indicates that TNF-alpha up-regulates ST 18 mRNA expression. The cells were also transfected with siRNA targeting ST18 to evaluate the effect of silencing of ST18 on the TNF-alpha-induced fibroblasts apoptosis. Apoptosis assay results showed that silencing ST18 resulted in 80% inhibition of TNF-alpha-induced apoptosis. Since ST18 is a transcription factor, the next step was to investigate whether ST18 plays a role in TNF-alpha-induced apoptotic and pro-inflammatory gene expression. Real-time PCR was performed on pro-apoptotic genes, TNF-alpha, caspase-3 and Fas, and pro-inflammatory gene IL-1 and IL-6 to evaluate the effect of silencing of ST 18 on the gene expression. TNF-alpha-stimulated mRNA levels of the pro-apoptotic genes, such as TNF-alpha, caspase-3 and Fas were reduced significantly by silencing of ST18 (P[less than]0.05). Regarding pro-inflammatory genes, IL-1 and IL-6 mRNA levels were down-regulated dramatically upon silencing of ST18. In order to further investigate the role of ST 18 plays in relation to TNF-alpha stimulation on other apoptotic and inflammatory genes, the microarray analysis was performed using an Affymetrix GeneChip Human Genome U133 plus 2.0 array. The RNA profiling analysis further confirmed our results: using 2 fold as the threshold and significance level at P[less than]0.05, the genes showed significantly activated/inactivated by silencing of ST18, including 193 apoptosis-related genes and 201 inflammation- related genes. In order to study the direct role that ST 18 played in TNF-alpha induced fibroblast apoptosis, we transiently transfected ST18 expression vector into the fibroblasts. The over-expression of ST18 caused a 2.9-fold increase in fibroblast apoptosis compared to the empty-vector-transfected group. Not only did over-expressed ST18 result in fibroblast apoptosis but it also up-regulated the mRNA levels of TNF-alpha, IL-1 and IL-6 significantly (p[less than]0.05). Using promoter-reporter system, we have also demonstrated the transcriptional activity of ST18 in activating transcription of TNF-alpha and IL-1. Taken together, there results indicated that ST 18 is a crucial signaling molecule in mediating the TNF-alpha-induced apoptosis and inflammatory gene expressions.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (D.Sc.D.)--Boston University, Goldman School of Dental Medicine, 2006 (Oral Biology).Includes bibliographical references: leaves 105-112.
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