Type 2 diabetes is a metabolic disorder characterized by increased glucose concentrations in the blood due to decreased insulin sensitivity. The worldwide incidence of diabetes has increased remarkably over the last two decades. Obesity, due to increased consumption of calorie dense diets, and sedentary life styles, is commonly cited as a primary cause. However, many epidemiological studies have established a relationship between insulin resistance and exposure to environmental chemicals such as persistent organic pollutants (POPs). The mechanisms by which POPs alter metabolism remain poorly understood, although their lipophilic nature suggests a role in adipose tissue function. The Tischkau lab has established a relationship between Aryl hydrocarbon Receptor (AhR) activation by different types of POPs and increased risk of insulin resistance. This dissertation, therefore, explored the effects of AhR activation by POPs on adipose tissue function. Adipose tissue regulates systemic glucose and lipid metabolism through production of hormones and cytokines that regulate appetite and energy homeostasis. It is well-known that impaired adipose function promotes systemic insulin resistance. The first specific aim examined the hypothesis that activation of AhR suppresses adipogenesis by lowering the rate of pre-adipocyte differentiation. Adipogenesis is a process by which mesenchymal stem cells (MSCs) and pre-adipocytes differentiate into mature adipocytes. Limitations in adipogenesis and accumulation of ectopic lipid have significant roles in decreasing insulin sensitivity. Thus, I hypothesized that POPs contribute to systemic insulin resistance by lowering the rate of MSCs and preadipocyte differentiation; the resulting large, poorly-functioning adipocytes increase serum lipids and promote lipid deposition in other tissues. MSCs derived from mouse bone marrow and pre-adipocytes were treated with different concentrations of AhR agonist, β-Naphthoflavone (BNF), and levels of transcripts associated with adipocyte differentiation were determined by using quantitative PCR. Oil red O staining and lipid content were observed to examine differentiation into mature adipocytes. Genes that promote adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), and adiponectin were downregulated in MSCs treated with BNF. Moreover, accumulation of triglycerides was decreased after BNF treatment. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on adipogenesis. Therefore, activation of AhR by exogenous ligands inhibits adipogenesis leading to impaired fat storage. Limitations in adipogenesis promotes accumulation of the excess lipid in non-fat tissue such as liver, muscle, and heart leading to decrease the insulin sensitivity and disrupt energy homeostasis. The second specific aim examined effects of AhR activation on circadian clock regulation in adipose tissue. A circadian clock essentially regulates systematic energy homeostasis; the central clock in the suprachiasmatic nucleus (SCN) works with the local clocks in peripheral tissues such as liver, muscle, and adipose tissue to regulate whole-body metabolism. The Tischkau lab has previously shown that AhR interacts with the core machinery of the circadian clock. Activation of AhR by environmental toxicants leads to a dampening of the rhythm expression of core clock genes or an alteration in the timing of their peak expression, which subsequently promotes metabolic disorders such as glucose insensitivity and hyperlipidemia. Given the importance of appropriately timed adipose tissue function to regulation of energy homeostasis, this study focused on mechanisms by which AhR may influence clock-controlled mature adipose tissue activity. Lipolysis is a clock-regulated process in adipose tissue that provides the necessary energy during periods of fasting and exercise. Thus, I hypothesized that AhR activation in adipose tissue would impair lipolysis by altering molecular circadian clock function. AhR activation was proposed to dampen adipose rhythms, leading to a decreased lipolysis rate during the absence of food, and subsequently, increased glucose concentrations in the blood. C57BL/6 mice were injected with vehicle or 50 mg/kg body weight of the AhR agonist, BNF, 48 hours after release into constant darkness. Mice were sacrificed, and epididymal adipose tissue was collected every 6 hours over a 24 hour period. Real-Time RT-qPCR was used to measure mRNA expression of genes responsible for lipolysis. To examine effects of AhR activation in vitro; mouse pre-adipocytes, 3T3-L1 cells, were differentiated into mature adipocytes for 12 days. Cells were then starved for 24 hours with DMEM media containing 1% FBS to induce lipolysis in the presence of 100, 200, 300 µM of BNF. RNA was then extracted and mRNA expression for genes responsible for circadian clock and lipolysis were determined by RT-qPCR. Alterations were observed in rhythms of core clock genes in wild type mice injected with BNF compared to wild type mice injected with vehicle. Rhythms of key enzymes controlling lipolysis including hormone sensitive lipase (HSL) and adipose triglycerides lipolysis (ATGL) was changed in wild type mice injected with BNF compared to wild type mice injected with vehicle. These effects were blocked in AhR deficient mice, suggesting that these effects were AhR dependent. Liver glycogen was decreased in mice injected with BNF compared to wild type mice injected with vehicle after 12 hour of food restriction but not in AhR null mice. Activation of AhR led to decreased expression of lipolysis genes in adipose tissue at CT6 (middle of the rest phase) as well as in 3T3-L1 cells. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on lipolysis in 3T3-L1 cell line. These data establish a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. The data from this study suggest that activation of AhR by BNF increases the risk of insulin resistance and type 2 diabetes by impairing adipogenesis. Reduced adipogenesis likely decreases adipocyte capacity to capture triglycerides from the blood. These effects may disturb energy homeostasis and contribute to the development of metabolic syndrome. This study also establishes a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. All together, these data suggest that environmental pollutants result in adipose tissue dysfunction by reducing adipogenesis and lipolysis. Therefore, activation of AHR by its exogenous ligands may increase the risk of insulin resistance and type 2 diabetes by impairing adipose tissue function. In particular, activation of AHR by exogenous ligands leads to impairment of free fatty acids storage during feeding and release during fasting to disturb energy homeostasis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24136号 / 医博第4876号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 波多野 悦朗, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Toxicity resulting from systemic administration continues to limit the effectiveness of modern chemotherapeutics. Administered drugs and biologics damage vital organs via off target effects while in circulation, decreasing the maximum tolerated dose and preventing the use of more effective drug concentrations. Many therapeutic agents are additionally constrained by a narrow therapeutic index, requiring delicate balance between toxicity and loss of efficacy. With dose remaining a critical determinant of efficacy, the inability to utilize increased drug concentrations due to toxicity prevents the full exploitation of the drug’s therapeutic effect. While exploitation of the enhanced permeability and retention effect has been somewhat successful in targeting nanoparticles to tumors via passive accumulation, elevated tumor pressure and poor drug penetrance prevent a significant improvement in therapeutic effect. Here we describe the conjugation of polymer prodrugs to tumor homing mesenchymal stem cells as a novel approach to chemotherapeutic drug delivery. The utilization of an actively homing cellular vehicle for effective drug delivery against the pressure gradient has the potential to improve drug penetration of the tumor while reducing systemic toxicity and improving drug efficacy.

Acute myeloid leukemia (AML), a blood malignancy resulting in abnormal hematopoiesis, is associated with alterations in the bone marrow environment (BME). Current treatments for this heterogeneous disease, mainly targeting the leukemic cells, are largely unsuccessful for the majority of AML subtypes. By better understanding the mechanisms by which the BME contributes to leukemogenesis, it may be possible to introduce more effective treatments for AML. Mesenchymal stromal/stem cells (MSCs) are essential cellular components of the BME/hematopoietic niche and have been shown to support normal hematopoiesis. As a critical component, they may have several roles in altering the BME, thus providing an excellent model for studying the BME in-vitro. Several studies have characterized AML-derived MSCs (AML-MSCs). However, their exact role in altering BME remains unclear. Here, we investigated the MSCs' potential role in BME alteration by investigating the genetic profiles of previously characterized AMLMSCs (n=29) and healthy donor MSCs (HD-MSCs) (n=8). We identified that among 7565 common genes, 21 genes were significantly differentially expressed in AMLMSCs. The CD248 gene was identified among these significantly upregulated genes in AML/HD-MSCs (n=29). Focusing on AML-MSCs derived from high-risk patients (HR), CD248 protein was investigated and validated using HR AML-MSCs (n=11) and HD-MSCs (n=4). Interestingly, it was highly abundant in HR samples at the intracellular and cell-surface levels. CD248 is an MSC marker and has a biological significance potentially on their function. To better understand its potential role in MSC, CD248 was knocked down (KD) in HD-MSCs using siRNA (siCD248-MSCs). Functional capacity, the ability of HD-MSCs and siCD248-MSCs to differentiate into cell types that form the BME (adipocytes and osteocytes), and their ability to promote the growth of HL60 human leukemia cell line were assessed. Posttransfection functional assessments showed that siCD248-MSCs had a reduced adipogenic but not osteogenic potential via differentiation assays. Quantitative validation of the adipogenesis pathway by qRT-PCR confirmed the reduction. KD CD248 increased SIRT2 expression and potentially led to adipogenesis inhibition. However, co-culture experiments showed no effect of HD-MSCs or siCD248-MSCs on HL60 proliferation. Together these data showed that CD248 is a potential player in adipogenesis, essential to MSC’s functionality. Thus, it could serve as a prognostic marker and target for AML therapy.

No description available.

Preterm birth (<37 weeks) is the world’s principal cause of death of children <5 years of age. Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth. BPD is characterized by an arrest in alveolar and vascular development within the lung. It is a multifactorial disease, caused by a combination of supplemental oxygen, mechanical ventilation, and inflammation. BPD is also an independent risk factor for abnormal neurodevelopment. The link between BPD and abnormal neurodevelopment is poorly understood and there are currently no effective cures for these complications. We hypothesized that a crucial cell population for brain development, i.e., the neural progenitor cell (NPC) is functionally impaired in BPD and that this impairment is associated with abnormal neurodevelopment. Based on our previous findings, we also predicted that human umbilical cord-mesenchymal stromal cell (UC-MSC) extracellular vesicles (EVs), could mitigate both the lung and brain injuries in experimental BPD. We utilized several animal models of BPD, across multiple species, to determine the effects of hyperoxia, mechanical ventilation, and inflammation on the developing lungs and brain. We also utilized UC- MSC therapy to mitigate these injuries. We discovered that hyperoxia exposure damages the developing lungs as well as the brain, leading to cerebrovascular and NPC impairments, as well as reduced neurogenesis. These impairments were associated with neurobehavioural deficits in adulthood. Furthermore, we found that inflammation in combination with mechanical ventilation and hyperoxia also impairs NPC function. Importantly, we demonstrated that UC-MSC EVs can reduce inflammation, improve vascular growth, restore lung growth, and mitigate impairments in NPC self-renewal. This work highlights novel mechanisms of BPD-associated abnormal neurodevelopment and offers potential regenerative medicine therapies to alleviate these outcomes for this vulnerable population.

Digital image processing is widely used in the field of time-lapse microscopy and biological research to provide statistical data of cellular dynamics. The data can provide more comprehensive understanding of the molecular phenomenon. Further, digital image processing enables rapid and consistent quantification of qualitative observations. The image processing model examined here provides a study to identify structures called retraction fibers (RFs) that are formed during epithelial-mesenchymal transition (EMT) [1], an important developmental process which also occurs during cancer metastasis. Quantifying RF formation is an important task for biologists studying cellular regulation of EMT. This thesis work uses digital image processing and computer vision algorithms to detect and track each RF in image sequences of cells undergoing EMT that are captured using time-lapse microscopy. The algorithms isolate the RFs with reasonable precision. Statistical information is generated about these automatically detected RFs, such as the number formed during a particular time window, lifetime of each, and their geometric dimension. This information can in turn be used by biologists to quantitatively measure the extent of EMT under different test conditions. Biologists feel that the information thus obtained may help clarify the molecular interactions of cell migration and will aid in developing methods of preventing cancer metastasis. Experimental results show that this methodology has significant potential in helping biologists determine RF behavior during EMT.

Development is punctuated by morphogenetic rearrangements of epithelial tissues, including complete detachment of individual motile cells during epithelial-mesenchymal transition (EMT). Dramatic actin rearrangements occur as cell-cell junctions are dismantled and cells become independently motile during EMT. Characterizing dynamic actin rearrangements and identifying actin machinery driving these rearrangements is essential for understanding basic mechanisms of cell-cell junction remodeling; yet, neither the precise series of actin rearrangements at cell-cell junctions that accompany EMT, nor the machinery that controls actin rearrangement during EMT, have been identified. This work represents a detailed study of junctional actin reorganization in cells undergoing EMT, identifies actin regulatory proteins that control this actin reorganization, and defines the specific function of one regulatory protein, zyxin, in EMT. Using immunofluorescence and live cell imaging of HGF induced scattering of MDCK cells, dynamic actin rearrangement events occurring during EMT are characterized. Junctional actin characteristic of cell-cell adherent cells is rearranged into contractile medial actin networks linked to the junctional membrane in the initial steps of cell scattering. This actin rearrangement is accompanied by dynamic redistribution of specific actin regulatory proteins, namely α-actinin and zyxin-VASP complexes. α-Actinin mediates higher order structure of junctional actin. Zyxin-VASP complexes mediate linkage of dynamic medial actin networks to adherens junction membranes. Zyxin regulation of actin-membrane linkage controls whether cell migration during EMT occurs independently in solitary cells or is coordinated through tissues. The functional analysis employed here uses novel, quantitative methods that define specific cellular EMT ‘phenotypes’ to reveal the precise role of zyxin in EMT. Constitutive active zyxin mutants exhibit persistent actin-membrane linkages and a scattering phenotype in which cells migrate without loss of cell-cell adhesion. Zyxin is proposed to regulate EMT progression by regulating disruption or maintenance of actin membrane linkages at cell-cell junctions. Zyxin alters the ability of cells to fully detach and migrate independently during EMT and may be an important regulator of morphogenetic plasticity.

Hepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and migrating as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-Met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. This study addresses this shortcoming in a number of ways.A high-throughput in vitro drug screen was employed to identify proteins necessary in this HGF-induced signaling. Cells were tested for reactivity to HGF stimulation in a Boyden chamber assay. This tactic yielded several small molecules that block HGF-induced scattering, including a calcium channel blocker. Patch clamping was used to determine the precise effect of HGF stimulation on Ca2+ signaling in MDCK II cells. Cell-attached patch clamping was employed to detect Ca2+ signaling patterns, and channel blockers were used in various combinations to deduce the identity of Ca2+ channels involved in EMT. The results of these experiments show that HGF stimulation results in sudden and transient increases in calcium channel influxes. These increases occur at predictable intervals and rely on proper tubulin polymerization to appear, as determined through the use of a tubulin polymerization inhibitor. Though multiple channels occur in the membranes of MDCK II cells, noticeably TRPV4 and TrpC6, it is TrpC6 that is specifically required for HGF-induced scattering. These HGF-induced calcium influxes through TrpC6 channels drive a transient increase in NFAT-dependent gene transcription which is required for HGF-induced EMT. This was determined through the use of luciferase-based NFAT reporter assays and confirmed through confocal immunofluorescence. Using a small-molecule inhibitor of WNK kinase, it was determined that loss of WNK kinase function is sufficient to prevent HGF-induced EMT. Furthermore, patch-clamp analysis demonstrated that WNK kinase significantly increases channel opening at the surface of MDCK cells, indicating a possible mechanism of action for c-Met inhibition, but leaving doubt as to whether WNK kinase is in fact normally involved in c-Met signaling, or whether it is simply permissive.

The inevitable progression of advanced prostate cancer to castration resistance, and ultimately to lethal metastatic disease, depends on primary or acquired resistance to conventional androgen-deprivation therapy (ADT) and accumulated resistance mechanisms to evade androgen receptor (AR) suppression. Whereas the canonical androgen/AR signaling axis maintains prostate cell growth, differentiation and survival, in prostate cancer cells, AR adaptations that arise in response to ADT are not singular, but diverse, and include gene amplification, mutation and even complete loss of receptor expression. Collectively, each of these AR adaptations contributes to a complex, heterogenous, ADT-resistant tumor that culminates in prostate tumor cells transitioning from epithelial to mesenchymal states (EMT) and the development of metastatic castration-resistant prostate cancer (mCRPC). Here, we examined prostate cancer cell lines that model common CRPC subtypes, each with different AR composition, and focused on novel regulators of tumor progression, the Bromodomain and ExtraTerminal (BET – BRD2, BRD3 and BRD4) family of proteins, to test the hypothesis that each BET family member regulates EMT and underlying characteristics such as cell motility and invasiveness. We systematically manipulated the BET proteins and found that BRD4 regulates cell migration and invasion across all models of CRPC, regardless of aggressiveness and AR status, whereas BRD2 and BRD3 only regulate cell migration and invasion in less aggressive models that retain AR expression or signaling. We determined that BRD4’s contribution to this process occurs through the transcriptional regulation of AHNAK, SNAI1 and SNAI2, which are EMT genes linked to promotion of metastasis in a diverse set of cancers. Furthermore, treatment of CRPC cell lines with low doses of MZ1, a small-molecule, BRD4-selective degrader, inhibits EMT and metastatic potential. Overall, these results reveal a novel, BRD4-regulated EMT gene signature that may be targetable to treat metastatic castration-resistant prostate cancer.