Regardless of the original cause and etiology, the progression of kidney disease follows a final common pathway associated with tubulointerstitial injury, in which proximal tubular epithelial cells (PTEC) are instrumental. Kidney injury molecule-1 (KIM-1) is an emerging biomarker of kidney tubular damage. It is markedly expressed and released into urine in various animal models and human kidney diseases. This study aimed to explore the underlying mechanism regulating the release of KIM-1 by PTEC. First, expression and release of KIM-1 by primary cultured human PTEC were examined. In quiescent PTEC, KIM-1 was detected at the plasma membrane and in the cytoplasm. A transwell system, in which PTEC were grown as monolayer on permeable membrane, was used to examine the polarized release of KIM-1. PTEC constitutively released KIM-1 from their apical surface, and the release was independent of gene expression or protein synthesis. The KIM-1 release process by PTEC was enhanced dose- and time-dependently by two important kidney injury mediators, human serum albumin (HSA) and tumor necrosis factor (TNF)-α, and was inhibited by the presence of broad-spectrum inhibitors of matrix metalloproteinases (MMP). Second, the potential sheddases responsible for KIM-1 shedding were identified by quantitative polymerase chain reaction (PCR) array system, in which the gene expression of a panel of MMP members was screened. The gene expression of MMP-3, MMP-7 and MMP-9 was up-regulated by PTEC under HSA or TNF-α activation. Blockade experiments with synthetic MMP inhibitors or MMP gene knockdown by small interfering RNA transfection, revealed that the constitutive or accelerated KIM-1 shedding was mediated by MMP-3, but not MMP-7 or MMP-9. The role of MMP-3 in KIM-1 shedding was further defined by additional data showing the enhanced MMP-3 synthesis by HSA- or TNF-α-stimulated PTEC, and the up-regulated KIM-1 shedding by PTEC following exogenous MMP-3 treatment. Third, the regulatory mechanism of MMP-3-mediated KIM-1 shedding was investigated. Treatment of PTEC with HSA or TNF-α up-regulated the reactive oxygen species (ROS) generation, and its kinetics ran parallel to the increase of KIM-1 shedding and MMP-3 synthesis. In addition, exogenous hydrogen peroxide dose-dependently induced KIM-1 shedding and MMP-3 synthesis, which were abolished by the presence of an oxidation inhibitor. These evidence suggest that ROS play an essential role in regulating the MMP-3-mediated KIM-1 shedding by PTEC. Finally, a mouse model of acute kidney injury induced by renal ischemia and reperfusion (I/R) was established to translate the in vitro findings. Reduced kidney function and increased urinary KIM-1 level were observed in mice after renal I/R treatment. Strikingly, the expression of MMP-3 and KIM-1 in the I/R treated mice was most profound in the S3 segments of the proximal tubules, where is the most susceptible area to oxidative stress. Taken together, these in vivo data have further strengthened the distinct roles of ROS and MMP-3 in KIM-1 shedding during PTEC injury. In conclusion, ROS generated by the injured PTEC activate MMP-3, which release the soluble KIM-1 through the ectodomain shedding process. / published_or_final_version / Medicine / Master / Master of Philosophy

Angiogenesis is one of the essential hallmarks of cancer, typically breast cancer. Signaling from VEGFR-2 is necessary for the execution of VEGF-induced proliferation, migration, and tube formation of cultured endothelial cells in vitro and the onset of angiogenesis on tumors in vivo. Ellagic acid is a naturally existing small molecular polyphenol widely found in fruits and vegetables. It was reported that ellagic aicd interfered with some angiogenesis-dependent pathologies. Yet the mechanisms involved were not fully understood. Thus we analyzed its anti-angiogenesis effects and mechanisms on human breast cancer utilizing in vitro and in vivo methodologies. Besides, the in silico analysis was carried out to further analyze the structure-based interaction between ellagic aicd and VEGFR-2. The influences of ellagic aicd on VEGF-induced endothelial cells were studied by proliferation, tube formation and migration in vitro experiments. Kinase activity assay and western blotting were utilized to explore the effects of ellagic aicd on VEGFR-2 induced signaling pathway. Organ-based chick aortic ring model, in vivo Chorioallantoic membrane model and in vivo breast cancer xenografts were built to determine the anti-angiogenesis effects of ellagic aicd. Besides, software LigandFit algorithm in Discovery Studio 2.1 (Accelrys Inc., San Diego, CA) was applied to further understand the structure-based interaction between ellagic aicd and VEGFR-2. We found that ellagic aicd impeded a series of VEGF-induced angiogenesis processes including proliferation, migration and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways including MAPK and PI3K/Akt on endothelial cells. Ellagic aicd also obviously inhibited sprouts formation from chicken aorta and neo-vessel formation in chick chorioallantoic membrane. The growth and the P-VEGFR2 expression in breast tumors treated with ellagic aicd were also significantly suppressed. In the molecular docking simulation experiment, the structure-based interaction of VEGFR-2 with ellagic acid was found to be stable conformation by hydrogen bonds within residues Lys866 and Glu883 as well as by π–π interactions within residue Phe1045 at ATP binding pocket of VEGFR-2 catalytic domain. Taken together, ellagic aicd could exert anti-angiogenesis effects via VEGFR-2 signaling pathway in breast cancer. / published_or_final_version / Chinese Medicine / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Esophageal cancer is the third most common digestive tract malignancy. Along with surgery, genotoxic drugs (e.g. cisplatin) and radiotherapy are the mainstays of treatment for this disease. Environmental factors and environmental stress-induced responses contribute to esophageal tumorigenesis and chemoresistance. Studying key molecules in stress-induced signal pathway can help unravel the underlying mechanisms and discover rational therapeutic targets. Cyclin D1 is DNA damage response protein. Genotoxic stress induces rapid cyclin D1 degradation and the molecules mediating this response are cell-type dependent. The first part of this study investigated the changes of cyclin D1 expression in response to genotoxic stress in immortalized esophageal epithelial cells, which are experimental models commonly used to study the early events of cancer development. The results showed that cyclin D1 underwent rapid proteasomal degradation before p53-induced p21 accumulation, which substantiates that cyclin D1 plays a role in eliciting cell cycle arrest very early in the DNA damage response. FBXO31 and FBX4, two F-box proteins previously reported to mediate cyclin D1 degradation, were found to be accumulated and unchanged, respectively, after ionizing irradiation in immortalized esophageal epithelial cells and esophageal squamous cell carcinoma (ESCC) cell lines. Yet, knockdown of FBXO31 did not rescue rapid cyclin D1 degradation upon UV or ionizing irradiation. This led to the hypothesis that accumulation of FBXO31 may have novel functions beyond mediating cyclin D1 degradation in cells responding to genotoxic stress. The second part of this study explored the function of FBXO31 in genotoxic stress response. The accumulation of FBXO31 in cancer cells after exposure to various genotoxic stresses was found to coincide with p38 deactivation, giving the clue that FBXO31 may negatively regulate this important pathway. Further studies revealed that knockdown of FBXO31 resulted in sustained activation of stress-activated MAPKs (SAPKs) p38 and JNK, as well as increase in UV-induced cell apoptosis, whereas overexpression of FBXO31 had opposite effects. The inhibitory role of FBXO31 on SAPK activation and apoptosis was confirmed by shRNA rescue experiments. Consistent with the observed anti-apoptotic effect, soft agar, colony formation and in vivo xenograft experiments showed that FBXO31 had oncogenic function in ESCC. Moreover, in vitro and in vivo results showed that knockdown of FBXO31 could sensitize ESCC cells to cisplatin treatment. The mechanism underlying the inhibition of SAPKs by FBXO31 was investigated in the third part of this study. Co-immunoprecipitation results showed that FBXO31 could interact with MKK6 (a p38 activator), but not p38, JNK1, or other MAP2Ks. FBXO31 was found to be co-localized with MKK6 in the cytoplasm. Mapping of interaction domains of FBXO31 revealed that aa 115-240 and aa 351-475 were responsible for binding to MKK6. Further study found that binding of FBXO31 to MKK6 could facilitate the K48-linked polyubiquitination and degradation of MKK6. Taken together, the results of this study showed that FBXO31 accumulation upon genotoxic stress can promote the degradation of MKK6 via K48-linked ubiquitination, thereby inhibiting SAPK activation and protecting cancer cells from genotoxic stress-induced apoptosis. FBXO31 may be a potentially useful therapeutic target to overcome chemoresistance in cancer therapy. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is the seventh most prevalent cancer and the third leading cause of cancer-related mortality globally. Metastasis is a major cause of mortality. HCC is also highly chemoresistant which limits treatment options to patients. Understanding the molecular mechanisms involved in these two events is of crucial significance. Deregulation of Rho/ROCK signaling is common in HCC and regulates different cellular events including cell invasion and survival. In this study, we aimed to further investigate how members of the Rho/ROCK pathway regulate HCC cell invasion and chemoresistance. By screening 71 pairs of human HCC samples using real-time qPCR, we identified that RhoE was frequently downregulated in human HCC. RhoE serves as an antagonist of the Rho/ROCK pathway. Clinicopathologically, downregulation of RhoE associated with shorter patient disease-free survival. In virto assays showed that stable knockdown of RhoE enhanced both HCC cell migration and invasion. In vivo mouse models also demonstrated that knockdown of RhoE promoted HCC invasiveness and intra-hepatic metastasis. Mechanically, knockdown of RhoE increased ROCK activity By screening 71 pairs of human HCC samples using real-time qPCR, we identified that RhoE was frequently downregulated in human HCC. RhoE serves as an antagonist of the Rho/ROCK pathway. Clinicopathologically, downregulation of RhoE associated with shorter patient disease-free survival. In virto assays showed that stable knockdown of RhoE enhanced both HCC cell migration and invasion. In vivo mouse models also demonstrated that knockdown of RhoE promoted HCC invasiveness and intra-hepatic metastasis. Mechanically, knockdown of RhoE increased ROCK activity and inhibition of ROCK reversed the effect of RhoE knockdown on cell migration. RhoE overexpression induced disassembly of stress fibers while knockdown of RhoE enhanced formation of plasma membrane blebs. These findings suggested that RhoE acts as a metastatic suppressor in HCC via inhibiting Rho/ROCK signaling. Downregulation of RhoE can increase ROCK activity which is reported to regulate cell survival. Therefore we investigated if the frequent downregulation of RhoE contributes to the high chemoresistance in HCC cells. Stable knockdown of RhoE suppressed cell death/apoptosis induced by chemotherapeutic agents such as cisplatin and doxorubicin. This effect could be reversed by addition of ROCK inhibitor. In vivo mouse model also confirmed that RhoE knockdown augmented HCC chemoresistance. We also observed that combined treatment of cisplatin and ROCK inhibitor profoundly inhibited tumor growth in nude mice. This part of our findings indicated that RhoE/ROCK played an important role in regulating chemoresistance in HCC. We further identified two downstream molecular pathways which were involved in Rho/ROCK-induced chemoresistance. We found that STAT3 and JAK2 were activated by RhoE knockdown but inhibited by addition of ROCK inhibitor. Upon ROCK inhibition, expression of IL-6 and IL-6 receptor were suppressed and the transcription activating activity of STAT3 was also repressed. Finally, ROCK inhibition attenuated Erk1/2 activation. Literature searching suggested nuclear PTEN as a potential candidate for inactivating Erk1/2. We demonstrated that inhibition of ROCK increased the population of nuclear PTEN while overexpressing ROCK2 decreased it. Overexpression of nuclear PTEN alone could already reduce Erk activation in HCC cells. Our findings indicated that RhoE/ROCK may exert their effects on chemoresistance in HCC via regulating the IL-6/JAK2/STAT3 and PTEN/Erk pathways. In conclusion, our study demonstrated the important role of RhoE in HCC. First, aberrant underexpression of RhoE promoted HCC invasion and intra-hepatic metastasis through upregulating the Rho/ROCK signaling. Second, downregulation of RhoE increased activity of the pro-survival IL-6/JAK2/STAT3 and Erk signalings to enhance chemoresistance in HCC cells. Our findings also suggested the Rho/ROCK signaling to be potential therapeutic target in anti-metastatic and chemo-sensitizing therapy. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Presenilin (PS) is the catalytic subunit of the gamma-secretase which is responsible for the cleavage of amyloid precursor protein to form beta amyloid (Aβ). Mutations in PS cause familial Alzheimer’s disease (FAD) by increasing the Aβ plaques formation in the brain and thereby induce neurodegeneration. Apart from this, FAD-linked PS mutations have been demonstrated to disrupt cellular calcium (Ca2+) homeostasis. Ca2+is a vital secondary messenger that involved in various neurophysiological functions, including memory, learning, and neuroplasticity and mounting evidence suggesting that Ca2+dysregulation associated with PS mutations may play a proximal role in the AD pathogenesis. Yet, the molecular mechanism for Ca2+dysregulation in AD remains debatable. It has been reported that cellular Ca2+homeostatsis can be disrupted in various ways. On the one hand, mutant PS has been demonstrated to exaggerate Ca2+release from the endoplasmic reticulum (ER) through different pathways. On the other hand, attenuatedCa2+influx from the extracellular medium through the capacitative Ca2+entry (CCE) pathway has also been reported to bring about cellular Ca2+disruption. However, the molecular mechanism for the PS mutation-mediated CCE deficits is largely unknown. For this reason, the objective of the current study is to elucidate the underlying molecular mechanism for attenuated CCE in AD. In this study, human neuronal cell line SH-SY5Y is employed as a cellular model to investigate the effect of wild-type or FAD-linked PS1 mutation on CCE pathway. Using single cell Ca2+imaging technique, significant CCE deficits was observed in SH-SY5Y stably expressing FAD-linked PS1mutation, PS1M146L. Interestingly, this CCE attenuation in PS1 mutant expressing cells was not mediated by the down-regulation of STIM1 and Orai1 expression, the known essential molecular players in the CCE pathway. Instead, co-immunoprecipitation and proximity ligation assay have suggested a physical interaction between PS1 and STIM1 proteins. Moreover, a putative gamma-secretase mediated STIM1 cleavage was discovered by western blotting. In addition, confocal imaging showed that PS1M146L significantlyreduceSTIM1 puncta formation and ER translocation followed by the activation of CCE pathway by ER Ca2+store depletion with thapsigargin. This indicated that mutant PS1 attenuates CCE by affecting STIM1 oligomerization or its recruitment with Orai1. Taken together, our results suggested the negative regulatory role of PS on CCE pathway and hypothesized the molecular mechanism of CCE where FAD-linked PS mutation is perceived as a gain-of-function mutation and enhanced the negative impact on STIM1 to inhibit Ca2+entry.This hypothetic model provides new insights into the molecular regulation for CCE pathway and the identification of the interacting domains between PS1 and STIM1 may suggest novel targets for the development of therapeutic agents that help to treat the disease. / published_or_final_version / Physiology / Master / Master of Philosophy

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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences