<p>Michelle N. Ingle. M.S., Purdue University, December 2019. Computational Modeling of Transforming Growth Factor-β2 Receptor Complex Assembly. Major Professor: David M. Umulis.</p> <p> </p> <p> Transforming growth factor (TGF)-β1, TGF-β2, and TGF-β3 are secreted signaling proteins that play an essential role in tissue development, immune response, and physiological homeostasis. TGF-β ligands signal through a tetrameric complex made up of two type I receptors (TβRI) and two type II receptors (TβRII). Dysregulation of TGF-β signaling has been linked to uncontrolled cell proliferation and cancer metastasis. An accurate understanding of TGF-β’s receptor complex assembly pathway may allow for pharmacological intervention and/or preservation of proper TGF-β signaling.</p> <p> Amongst the ligand types, TGF-β1 and TGF-β3 are efficient signalers, presumably by strong binding to both type I and II receptors. However, TGF-β2 has a very weak affinity for TβRII and requires an additional membrane-bound protein called betaglycan (BG) to achieve similar levels of downstream signaling. While computational modeling has been performed on the signaling pathway of the TGF-β system, to date no computational modeling has aimed to decipher BG’s role in the potentiation of TGF-β2 signal. To determine the role of BG in selectively facilitating signaling by TGF-β2, we developed computational models with different assumptions based on the levels of cooperativity between receptor subtypes and types of BG behavior (No Receptor Recruitment model, Single-stage Receptor Recruitment model, and Two-stage Receptor Recruitment model). </p> <p> With each of the receptor recruitment models we hypothesized that BG uses two domains to successfully enhance TGF-β2 signaling. This model was first proposed in Villarreal et al., 2016 and is further investigated in this work using a two-step computational approach. First, a root mean square error (RMSE) calculation was performed between our computational models with no BG present and published experimental signaling data in cell lines with no BG present. Lower RMSE values indicate the simulated data is more representative of experimental signaling behavior when no BG is present. The second round of model validation was performed by adding BG into the simulations and comparing its behavior to experimentally determined and hypothesized behaviors of BG. </p> <p> In summary, the simulations indicate there may be more cooperative receptor recruitment present in the system then stated in literature. Furthermore, it appears that BG binding to TGF-β2 ligand through two domains provides an effective transfer mechanism that can be tuned to control differential signaling between TGF-β ligand subtypes. Experiments were then suggested in order to support or refute one of the models offered in this thesis. For the purpose of uncovering how BG enhances TGF-β2 signaling, the computational work performed in this thesis highlights the areas where researchers should focus their experimental efforts and provides a baseline model for further computational work in the TGF-β system.</p>

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Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Transforming growth factor β1 (TGF-β1) is an abundant growth factor that is known to stimulate bone formation. This study was designed to examine the role of TGF-β1 on TNF-α-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 20 ng/ml of TNF-α, 10 ng/ml of TGF-β1, or combination, for 6 h. TNF-α exerted a variety of effects on the apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that TNF-α upregulated the mRNA levels of caspase-1, -7, -11, -12, and FAS. Western blot analysis showed enhanced processing of caspase-1, -7, -11, and -12, with the appearance of their activated enzymes 24 h after TNF-α treatment. In addition, caspase-3-like activity was significantly activated following TNF-α treatment. Levels of cleaved poly(ADP-ribose) polymerase and FAS protein were also elevated by TNF-α. Finally, Hoechst staining, terminal deoxynucleotidyl-transferase nick-end labeling (TUNEL) assay, and oligonucleosome ELISA all indicated that TNF-α induced apoptosis. In contrast, the addition of TGF-β1 attenuated all of the aforementioned effects of TNF-α. Our results demonstrate that TGF-β1 can decrease TNF-α-induced apoptosis in murine osteoblasts at least in part by attenuating TNF-α-induced caspase gene expression.

Depending on the duration and severity, psychological tension and physical stress can enhance or suppress the immune system in both humans and animals. Although it has been established that chronic stress exerts a significant suppressive effect on immune function, the mechanisms by which affects immune responses remain elusive. By employing an in vivo murine system, we revealed that TGF-β1/Smad2/3/Foxp3 axis was remarkably activated following chronic stress. Furthermore, TLR9 and p38 MAPK played a critical role in the activation of TGF-β1/Smad2/3/Foxp3 signaling cascade. Moreover, inhibition of TGF-β1/Smad2/3/Foxp3 or p38 significantly attenuated chronic stress-induced lymphocyte apoptosis and apoptosis-related proteins, as well as the differentiation of T regulatory cells in spleen. Interestingly, disequilibrium of pro-inflammatory and anti-inflammatory cytokines balance caused by chronic stress was also rescued by blocking TGF-β1/Smad2/3/Foxp3 axis. These findings yield insight into a novel mechanism by which chronic stress modulates immune functions and identifies new targets for the development of novel anti-immune suppressant medications.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19396号 / 医博第4047号 / 新制||医||1012(附属図書館) / 32421 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 野田 亮, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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This thesis will be organized into three chapters discussing the mechanism underlying the onset and progression of osteoarthritis (OA) in the temporomandibular joint (TMJ). Understanding the mechanism of OA development in the TMJ helps in understanding how OA progresses and how to treat this disease. The goal of this investigation is to examine the process of cartilage degeneration and OA biomarker expression in the TMJ to understand their role in TMJ OA onset and development.Chapter one covers mechanisms that are altered in TMJ OA during disease progression. Using animal models with different stressors such as mechanical disturbances, direct injury, and changes in the extracellular matrix composition revealed the role of the different mechanisms that are up-regulated and down regulated during cartilage destruction. Chapter two will cover a paper I wrote that introduces a novel non-invasive technique applied to mice, which induces an early onset of OA in the TMJ. I developed this technique with the aim to provide a new mouse model where the onset and progression of OA more closely mimic the natural TMJ OA progression in humans. The histopathological analysis of the cartilage demonstrates that onset of OA starts at 2 weeks after treatment induction and is aggravated by week eight. This data demonstrated the effectiveness of our technique in inducing OA in the TMJ. Chapter three will cover a second paper I wrote on the association of RAGE with the progression of OA in the TMJ of mice by using mice with and without RAGE expression. RAGE has been show to contribute to the progression of OA by releasing several pro-inflammatory and catalytic cytokines. Additionally, RAGE has been shown to modulate the expression of specific OA biomarkers, including HtrA-1, Mmp-13, and Tgf-β1 in knee cartilage. The objective of this study was to study the effect of knocking out RAGE on the expression of Mmp-1 3, HtrA-1, and Tgf-β1 in the TMJ. After histophatological and quantitative analysis of biomarkers expression, the results demonstrated for the first time that absence of RAGE expression in the TMJ provides a protective effect against development of TMJ OA in mice.

Atherosclerosis, is a chronic inflammatory disease, characterized by the narrowing of the arteries resulting from the formation of intimal plaques in the wall of arteries. Yet the molecular mechanisms responsible for maintaining the development and progression of atherosclerotic lesions have not been fully defined. In this study, we show that TGF-β activates the endothelial-to-mesenchymal transition (EndMT) in cultured human aortic endothelial cells (HAECs) and this transition is dependent on the key executor of the Wnt signaling pathway . This study presents the first evidence describing the mechanistic details of the TGF-β-induced EndMT signaling pathway in HAECs by documenting the cellular transition to the mesenchymal phenotype including the expression of mesenchymal markers α-SMA and PDGFRα, and the loss of endothelial markers including VE-cadherin and CD31. Furthermore, a short hairpin RNA (shRNA) screening revealed that Wnt2 signaling is required for TGF-β-mediated EndMT of HAECs. Also, we found that LDLR mice fed on a high-fat western-type diet (21% fat, 0.2% cholesterol) expressed high levels of Wnt2 protein in atherosclerotic lesions, confirming that this signaling pathway is involved in atherosclerosis . These findings suggest that Wnt2 may contribute to atherosclerotic plaque development and this study will render Wnt2 as a potential target for therapeutic intervention aiming at controlling atherosclerosis.

Thirty five million people are currently living with HIV-1 today with women accounting for half of infected individuals globally. Sexual transmission is the main route of HIV transmission with approximately 40% of HIV infections occurring when the mucosal lining of the female genital tract (FGT) is exposed to HIV in semen from an infected male partner. Seminal plasma (SP), the fluid portion of semen, is a complex fluid which plays an immunomodulatory role in the FGT for successful conception, largely due to its high concentrations of TGF-β1. Several factors in SP from HIV-uninfected men have been shown to either inhibit or enhance HIV infection in target cells, however it is not clear how SP from HIV infected men would modulate genital epithelial cells (GECs), the first cells that encounter HIV in the FGT. The overall goals of this thesis were to compare inflammatory and regulatory cytokine concentrations in SP from HIV-uninfected and infected men, and subsequently compare GEC cytokine responses following exposure to SP from HIV-uninfected and HIV-infected men. I also investigated how SP and TGF-β regulated cytokine production and barrier function in GECs in the presence of HIV. The results summarized in this thesis demonstrated that HIV infection leads to different cytokine profiles in SP, based on stage of HIV-1 infection. HIV-infected men in acute stage contained higher levels of proinflammatory cytokines in their SP compared to HIV-uninfected and chronically infected men (CI men) which subsequently lead to higher levels of proinflammatory cytokines from GECs compared to CI men. In the follow up to this study we found that active TGF-β, which was found in higher concentrations in SP from CI men and led to decreased inflammatory response from GECs, was compartmentalized between blood plasma and seminal plasma. Higher levels of active TGF-β in SP correlated with decreased semen viral load and the immune activation marker sCD14 leading us to believe that ART-naive CI men in our cohort were naturally controlling their immune activation status, as active TGF-β levels were lower in ART-treated men. Short-term exposure of GECs to SP from CI men or TGF-β at comparable concentrations to SP protected the GEC barrier against HIV by decreasing inflammatory cytokines and preventing tight junction breakage. However, long-term exposure to TGF-β in the presence of HIV further increased inflammation in GECs suggesting a biphasic role for TGF-β in the FGT. This body of work summarized in this thesis demonstrates for the first time how semen from HIV-infected men modulates FGT epithelial cell cytokine responses and barrier function, an important consideration in the design of local therapeutic strategies to protect the FGT against HIV infection. / Thesis / Doctor of Philosophy (Medical Science)