An Investigation of the Function of IκB Family Member Bcl-3 and its Role in Oncogenesis

Bcl-3 was originally identified as a protein that is highly expressed in certain B-cell lymphomas harboring a 14;19 translocation, and more recent evidence indicates that its expression is characteristic of a large number of human cancers. A member of the IκB family of NF-κB inhibitors, Bcl-3 interacts with NF-κB dimers containing p50 or p52 and promotes transcription of a subset of NF-κB dependent genes. However, neither the role of Bcl-3 in oncogenesis, nor its role in normal physiology, has been clearly defined. My research has focused on elucidating the function of Bcl-3 in the context of DNA damage signaling in order to gain insight into its physiological and pathological roles. Experiments presented here indicate that Bcl-3 is inducible by DNA damage and is required for the induction of Hdm2 gene expression and the suppression of persistent p53 activity. Furthermore, constitutive expression of Bcl-3 suppresses DNA damage-induced p53 activation and inhibits p53-induced apoptosis through a mechanism that is at least partly dependent on the upregulation of Hdm2. The results provide insight into a mechanism whereby altered expression of Bcl-3 leads to tumorigenic potential. Since Bcl-3 is required for germinal center formation, these results further indicate that Bcl-3 may play a critical in the B-cell maturation process by suppressing p53-dependent apoptosis in response to somatic hypermutation and class switch recombination. As aberrant expression of Bcl-3 has been found in an increasing number of human cancers, my second project set out to investigate whether Bcl-3 plays a role in hepatocellular carcinoma (HCC). The data indicate that nuclear staining of Bcl-3 is present in a high percentage of primary HCC tumors compared to adjacent normal tissue. Furthermore, western blot analysis indicates that high Bcl-3 expression correlates positively with expression of Hdm-2. These data suggest that Bcl-3 may play a causal role in HCC and indicate that it may be functioning through inhibition of p53. Collectively, the work described here proposes a novel function for the oncoprotein Bcl-3 that provides insight into both its normal and oncogenic roles and provides further evidence for the expanding role of Bcl-3 in human cancer

Expression of the Bcl-3 proto-oncogene suppresses p53 activation

While Bcl-3 expression in cancer was originally thought to be limited to B-cell lymphomas with a 14;19 chromosomal translocation, more recent evidence indicates that expression of this presumptive oncoprotein is significantly more widespread in cancer. However, an oncogenic role for Bcl-3 has not been clearly identified. Experiments presented here indicate that Bcl-3 is inducible by DNA damage and is required for the induction of Hdm2 gene expression and the suppression of persistent p53 activity. Furthermore, constitutive expression of Bcl-3 suppresses DNA damage-induced p53 activation and inhibits p53-induced apoptosis through a mechanism that is at least partly dependent on the up-regulation of Hdm2. The results provide insight into a mechanism whereby altered expression of Bcl-3 leads to tumorigenic potential. Since Bcl-3 is required for germinal center formation, these results suggest functional similarities with the unrelated Bcl-6 oncoprotein in suppressing potential p53-dependent cell cycle arrest and apoptosis in response to somatic hypermutation and class switch recombination

Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging.

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment

RALA and RALBP1 regulate mitochondrial fission at mitosis

Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission1. Equal distribution of mitochondria to daughter cells during mitosis requires fission2. Mitotic mitochondrial fission depends upon both the relocalization of large GTPase Drp1 to the outer mitochondrial membrane and phosphorylation of S616 on Drp1 by the mitotic kinase cyclin B/Cdk12. We now report that these processes are mediated by the small Ras-like GTPase RalA and its effector RalBP1 (RLIP76/RLIP1/RIP1)3,4. Specifically, the mitotic kinase Aurora A phosphorylates S194 of RalA, relocalizing it to the mitochondria, where it concentrates RalBP1 and Drp1. Furthermore, RalBP1 associates with cyclin B/Cdk1 kinase activity to foster phosphorylation of Drp1 on S616. Disrupting either RalA or RalBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B/Cdk1 converge upon RalA and RalBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function

NF-κB and IκBα are found in the mitochondria. Evidence for regulation of mitochondrial gene expression by NF-κB

The transcription factor NF-κB has been shown to be predominantly cytoplasmically localized in the absence of an inductive signal. Stimulation of cells with inflammatory cytokines such as tumor necrosis factor α or interleukin-1 induces the degradation of IκB, the inhibitor of NF-κB, allowing nuclear accumulation of NF-κB and regulation of specific gene expression. The degradation of IκB is controlled initially by phosphorylation induced by the IκB kinase, which leads to ubiquitination and subsequent proteolysis of the inhibitor by the proteasome. We report here that NF-κB and IκBα (but not IκBβ) are also localized in the mitochondria. Stimulation of cells with tumor necrosis factor α leads to the phosphorylation of mitochondrial IκBα and its subsequent degradation by a nonproteasome-dependent pathway. Interestingly, expression of the mitochondrially encoded cytochrome c oxidase III and cytochrome b mRNAs were reduced by cytokine treatment of cells. Inhibition of activation of mitochondrial NF-κB by expression of the superrepressor form of IκBα inhibited the loss of expression of both cytochrome c oxidase III and cytochrome b mRNA. These data indicate that the NF-κB regulatory pathway exists in mitochondria and that NF-κB can negatively regulate mitochondrial mRNA expression

Aurora-A Phosphorylates, Activates, and Relocalizes the Small GTPase RalA

The small GTPase Ras, which transmits extracellular signals to the cell, and the kinase Aurora-A, which promotes proper mitosis, can both be inappropriately activated in human tumors. Here, we show that Aurora-A in conjunction with oncogenic Ras enhances transformed cell growth. Furthermore, such transformation and in some cases also tumorigenesis depend upon S194 of RalA, a known Aurora-A phosphorylation site. Aurora-A promotes not only RalA activation but also translocation from the plasma membrane and activation of the effector protein RalBP1. Taken together, these data suggest that Aurora-A may converge upon oncogenic Ras signaling through RalA

Rosiglitazone Induces Mitochondrial Biogenesis in Differentiated Murine 3T3-L1 and C3H/10T1/2 Adipocytes

Growing evidence indicates that PPARγ agonists, including rosiglitazone (RSG), induce adipose mitochondrial biogenesis. By systematically analyzing mitochondrial gene expression in two common murine adipocyte models, the current study aimed to further establish the direct role of RSG and capture temporal changes in gene transcription. Microarray profiling revealed that in fully differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with RSG or DMSO vehicle for 1, 2, 4, 7, 24, and 48 hrs, RSG overwhelmingly increased mitochondrial gene transcripts time dependently. The timing of the increases was consistent with the cascade of organelle biogenesis, that is, initiated by induction of transcription factor(s), followed by increases in the biosynthesis machinery, and then by increases in functional components. The transcriptional increases were further validated by increased mitochondrial staining, citrate synthase activity, and O2 consumption, and were found to be associated with increased adiponectin secretion. The work provided further insight on the mechanism of PPARγ-induced mitochondrial biogenesis in differentiated adipocytes

Hypoxia-dependent mitochondrial fission regulates endothelial progenitor cell migration, invasion, and tube formation

Tumor undergo uncontrolled, excessive proliferation leads to hypoxic microenvironment. To fulfill their demand for nutrient, and oxygen, tumor angiogenesis is required. Endothelial progenitor cells (EPCs) have been known to the main source of angiogenesis because of their potential to differentiation into endothelial cells. Therefore, understanding the mechanism of EPC-mediated angiogenesis in hypoxia is critical for development of cancer therapy. Recently, mitochondrial dynamics has emerged as a critical mechanism for cellular function and differentiation under hypoxic conditions. However, the role of mitochondrial dynamics in hypoxia-induced angiogenesis remains to be elucidated. In this study, we demonstrated that hypoxia-induced mitochondrial fission accelerates EPCs bioactivities. We first investigated the effect of hypoxia on EPC-mediated angiogenesis. Cell migration, invasion, and tube formation was significantly increased under hypoxic conditions; expression of EPC surface markers was unchanged. And mitochondrial fission was induced by hypoxia time-dependent manner. We found that hypoxia-induced mitochondrial fission was triggered by dynamin-related protein Drp1, specifically, phosphorylated DRP1 at Ser637, a suppression marker for mitochondrial fission, was impaired in hypoxia time-dependent manner. To confirm the role of DRP1 in EPC-mediated angiogenesis, we analyzed cell bioactivities using Mdivi-1, a selective DRP1 inhibitor, and DRP1 siRNA. DRP1 silencing or Mdivi-1 treatment dramatically reduced cell migration, invasion, and tube formation in EPCs, but the expression of EPC surface markers was unchanged. In conclusion, we uncovered a novel role of mitochondrial fission in hypoxia-induced angiogenesis. Therefore, we suggest that specific modulation of DRP1-mediated mitochondrial dynamics may be a potential therapeutic strategy in EPC-mediated tumor angiogenesis