Keywords

Apoptosis, Bioinformatics, Cellular signal transduction, Gene expression, Interleukins, T cells, Autophagy, Glucose, Hexokinase, Interleukin 7, Lysosomes, Protein isoforms, Transcription Factor AP 1

Abstract

Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal transducers of the IL-7 growth signal: BimL and JunD. IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. After a bioinformatics survey to reveal possible JunD-regulated genes activated early in the IL-7 signaling cascade, our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism, including Pim-1. Pim-1, an IL-7 induced protein, was inhibited upon JNK or JunD inhibition. Our hypothesis that JunD positively regulated proliferation was confirmed when the proliferation of primary CD8+ T-cells cultured with IL-7 was impaired upon treatment with JunD siRNA. These results show that the IL-7 signal is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth through the expression of metabolic factors like HXKII and Pim-1. When metabolic activities are inhibited, cells undergo autophagy, or cell scavenging, to provide essential nutrients. Pro-apoptotic Bim was evaluated for its involvement in autophagy. Bim is a BH3-only member of the Bcl-2 family that contributes to T-cell death. Partial rescue of iv T-cells occurs when Bim and the interleukin-7 receptor are deleted, implicating Bim in IL-7- deprived T-cell apoptosis. Alternative splicing results in three different isoforms: BimEL, BimL, and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on IL-7 for growth, were used. Loss of Bim in IL-7-deprived T-cells delayed apoptosis, but blocked the degradative phase of autophagy. The conversion of LC3-I to LC3-II was observed in Bim-deficient T-cells, but p62, which is degraded in autolysosomes, accumulated. To explain this, BimL, was found to support acidification of lysosomes associated with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon IL-7 withdrawal only in Bimcontaining T-cells, indicating that in these cells autophagy was protective. IL-7 dependent Tcells lacking Bim were insensitive to inhibition of autophagy or lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosometracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, associated with intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of functional autolysosomes during autophagy

Notes

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Graduation Date

2012

Semester

Summer

Advisor

Khaled, Annette

Degree

Doctor of Philosophy (Ph.D.)

College

College of Medicine

Department

Molecular Biology and Microbiology

Degree Program

Biomedical Sciences

Format

application/pdf

Identifier

CFE0004435

URL

http://purl.fcla.edu/fcla/etd/CFE0004435

Language

English

Release Date

August 2013

Length of Campus-only Access

1 year

Access Status

Doctoral Dissertation (Open Access)

Subjects

Dissertations, Academic -- Medicine,Medicine -- Dissertations, Academic

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