| Eukaryotic cytoskeleton, a vital network system composed of microtubules, microfilaments and intermediate filaments(IFs), is another essential structural system apart from endomembrane and genetic systems. Not only is it involved in maintaining cellular morphology and the order of the intracellular structure, but also closely related to important cellular activities such as cytoarchitecture, differentiation, material transportation, signal transduction, gene expression, and cellular motility. Among these three kinds of cytoskeletons, IF superfamily are the most special in the following three aspects: 1. The basic components of the microfilaments and microtubules are limited to actin and tubulin respectively. However, the intermediate filaments can be formed by multiple subunits. 2. IF is the most stable cytoskeleton as well as the most complicated fiber structure in chemical composition among three kinds of cytoskeletons. 3. As important housekeeping proteins, microtubules and microfilaments play important roles in maintaining cellular structure and function during the whole cell fate. In contrast, IF is only expressed in special tissue in a specific temporal manner. Thus, most scholars believe that the biological function of the intermediate filament protein is not far limited to a kind of cytoskeletal protein. And further study on its structure and function will provide a crucial basis for us to analyze the characteristics of different sources and different stages of tissue cells.In vertebrates, intermediate filament protein family are divided into six categories based on their gene structure and expression patterns, such as acidic keratin(I), basic keratin(II), vimentin and desmin(III), neurofilament(IV), lamin(V), nestin(VI), etc. Although species of intermediate filament protein are diverse, their structures possess certain common points as follows: 1. All intermediate filament proteins consist of three parts: N-terminal, ?-helix domain and C-terminal tail domain. 2. They all have a characteristic central rod domain of about 310 amino acids capable of forming ?-helical coiled-coil dimers.3. Both head and tail domains are non-helical and exhibit huge variations in terms of size and amino acid composition.Previous studies have shown that different domains of intermediate filament protein are closely related to their functional characteristics. For example, N-terminal and C-terminal of Lamins are involved in its assembly process. Besides, the nuclear localization sequence between rod domain and C-terminal can guide Lamins into nucleus. What's more, keratin 18(K18) can regulate mitosis of hepatocytes via its N-terminal binging with 14-3-3, in which Ser33 phosphorylation of K18 plays an important role. Our team is committed to clarify the molecular mechanisms of intermediate filament protein Nestin in maintaining intracellular homeostasis and the role of Nestin+ cells in protecting tissue homeostasis and diseases treatment. Due to its long N-terminal and short C- terminal, Nestin is assembled into heterodimer with other intermediate filament proteins, like vimentin and ?-internexin, rather than in a homodimer manner by itself. Gene of Nestin in mice consists of four exons and three introns, and has three transcription initiation sites, on which transcription factor Sp1 and Sp3 can activate Nestin transcription. The first intron has muscle cell-specific enhancer activity and the second intron regulates its expression in neural progenitor cells of central nervous system. During embryonic development, Nestin is first expressed in neuroepithelial stem cells. As the cells undergoing differentiation, Nestin expression is down-regulated and disappeard after completely differentiation. Besides, Nestin is detected in endothelial cells of embryonic neovasculture, which is also down-regulated during the maturation of vessels. Thus, these Nestin+ cells are considered as an important part of adult stem cell pool, and play a significant role in maintaining stem cells homeostasis.Recently, using Nestin-GFP transgenic mice, Mendez-Ferrer et al firstly confirmed that there was a group of Nestin+ cells in bone marrow. They can form cloning and have abilities to osteogenesis, chrondrogenesis and adipogenesis, which indicates Nestin is an important candidate marker for bone marrow mesenchymal stem cells(MSCs). Subsequently, this team further proved that Nestin+ cells are divided into two subgroup, Nestinbright + and Nestindim + cells. It suggests that Nestin is not only a crucial marker of MSCs from bone marrow, but also participates in maintenance of bone marrow hematopoietic microenvironment. Interestingly, these discoveries lead to a question that is Nestin a common marker for MSCs from other tissues? Previously, our team had successfully isolated Nestin+ cells with self-renewal and multi-directional differentiation capacities from many tissues like bone marrow, testis and kidney from Nestin-GFP transgenic mice, which proved that Nestin is a specific marker of the adult MSCs. These results provided an important basis for further obtaining the MSCs for the purpose of damage repairing. On the other hand, we first constructed Nestin gene knockout mice models on the international, and found that Nestin gene deletion could cause a significant decline in self-renewal capacity and increase in death of neural stem cells. Meanwhile, Nestin knockout mice died at 10.5 days-11.5 days in embryonic stage, mainly results from the retardation of embryonic yolk sac vascular development and the death of perivascular cells with MSCs. Consequently, we can see that Nestin may be involved in self-renewal and maintenance of MSCs. And from above study, Nestin not only can be used as a marker for tissue mesenchymal stem cells, but also may serve as an important functional protein involved in the maintenance of stem cell pools in different tissues.In addition to the normal condition, Nestin is also expressed in abnormal tissue cells, including some of the damaged or diseased tissues. For example, glial cells and ventricular ependymal cells after spinal cord injury, astrocytes in retinal nerve injury, Nestin is re-expressed in the injury site, suggesting that Nestin may be involved in tissue repairing processes. Besides, Nestin was also found to be expressed in hepatic stellate cells. During hepatic fibrosis, hepatic stellate cells were activated and proliferated. At the same time, the expression of Nestin in hepatic stellate cells was increased, which suggested that it might be involved in the process of liver fibrosis. And recent studies have found that expression of Nestin is also detected in a variety of cancer tissues, such as brain glioma, bladder cancer, liver cancer, breast cancer, lung cancer and pancreatic cancer. Importantly, Nestin expression is positively correlated with the malignant degree of cancer, proliferation, metastasis. Therefore, Nestin is also considered to be a marker of various cancer stem cells. Our previous work used non-small-cell lung cancer cells as a model to explore the role and mechanism of Nestin in non-small cell lung cancer and found that expression of Nestin is related to prognosis of lung cancer. Then, we constructed sh Nestin A549, H460 and H1299 cell lines and found that proliferation ability decrease of cell after Nesin interfered. Further study showed that cell cycle was arrested in S phase and phosphorylation of Rb protein, phosphorylated Akt and phosphorylated GSK3?/? decreased, suggesting that Nestin is involved in regulation of non-small cell lung cancer cell proliferation through Akt-GSK3?/?-Rb signaling pathway. Thus, research about Nestin mainly focus on stem cells, damage repair and cancer models, and we can better understand its important role in intracellular homeostasis maintenance by studying biological function of Nestin in different cell types.The existing data shows that IF, as the major component of the cytoskeleton, may functions specifically to cellular stress by maintaining the intracellular homeostasis. 1. Apoptotic Stress. Apoptosis is a kind of basic physiological mechanism to maintain self-homeostasis. When suffering from extreme circumstantial stress, some damaged cells may sacrifice themselves for the whole organism. Multiple kinds of IF in various cells are able to protect cells from apoptosis. For example, Keratin 8 and Keratin 18 can bind to TRADD, the death-related domain of TNFR2 and TNFR, change the amount of Fas, thus protecting cells from apoptosis. In addition, knockout of Desmin in murine cardiomyocyte can induce apoptosis, which can be reversed by overexpression of anti-apoptotic protein Bcl-2. 2. Oxidative Stress. Reactive oxygen species(ROS) is generated in the process of metabolism or from external stimulation. It's a kind of oxygen-containing free radical and more reactive than molecular oxygen. Within the cell, ROS has two main resources, mitochondria and Nox family, which is involved in maintaining the normal function. However, the aberrantly accumulated ROS will trigger oxidative stress and even damage the cells. At that point, multiple antioxidants are needed to defense, while IF is supposed be a sort of antioxidant protein. Malhas et.al demonstrated that, the level of ROS increased as knockout of Lamin B1 in murine fibroblasts, which resulted in the increasing sensitivity to hydrogen peroxide. Meanwhile, knockout of Nestin in rat neural progenitor cells would promote apoptosis induced by hydrogen peroxide by unknown mechanism. 3. Metabolic stress. It's reported that there exists some links between IF and metabolism. In the meantime, multiple kinds of organelles are involved in the process of metabolism, especially the mitochondria. In a study by Tao Tang et.al, they found that knockout of Keratin 8 in the murine hepatocyte would cause significant collapse of the mitochondria, thus change in the metabolic function. However, how does IF target the organelles and what about the underlying mechanisms? Is Nestin involved in the regulation of organelle structure and function? Besides, what's the concrete mechanism of regulation of homeostasis by Nestin through ROS? All above require further exploration in the following days.Our lab have investigated the function of Nestin protein for a long time and found that Nestin played an important role in embryonic development in our previous research. Nestin knockout(KO) mice were lethal in the early period of embryonic development. Consistent with the phenomena observed above, a mass of Nestin KO embryonic stem cells(ESCs) underwent cell death during differentiating into embryonic body(EB) in vitro. Next, using several different inhibitors of programming cell death, such as Z-VAD-FMK(inhibitor of apoptosis), 3-Methyladenine(inhibitor of programming autophagy) and Nec1(inhibitor of necroptosis), we found that inhibitors of apoptosis and autophagy could not significantly revert the death of Nestin KO EB, whereas inhibitor of programming cell necrosis promoted Nestin KO EB survival. Furthermore, the main mechanisms of programming cell necrosis included RIP1/RIP3 complex elevate the general levels of energy metabolism and mitochondrial ROS generation, then disrupting cellular homeostasis and resulting in cell death. These findings demonstrated that the main reason of programming cell necrosis was increased intracellular ROS generation during the progression of Nestin KO ESCs differentiating into EB. Moreover, the cell death caused by Nestin deletion could be reverted by reducing intracellular ROS levels using ROS scavenger NAC. However, the molecular mechanisms by which IF Nesitn influenced intracellular ROS levels and regulated redox homeostasis remained unclear.Using assays of Co IP combined with mass spectrometry and super-resolution optical microscope, we found that Nestin was bound and interacted with several proteins, such as heat shock protein(HSP) family, endoplasmic reticulum calcium ATPase(SERCA2), nuclear lamina protein(Lamin A/C) and so on. Interestingly, most proteins were localized on endomembrane system using further bioinformatics analysis, which dedicated that Nestin participated in maintaining stability of endomembrane system. Since Nesitn could bind with these multi-functional proteins, we asked that whether these proteins took a role in Nestin targeting to endomembrane system, whether the interaction was associated with stability maintenance of endomembrane system? Meanwhile, the molecular mechanisms in Nestin regulating intracellular ROS levels remain unknown. Whether reducing ROS generation via modulating mitochondrial metabolism, or increasing elimination capacities via influencing intracellular antioxidant system needed to be further investigated. In all, these observations implied that intermediate filament proteins were closely related with cellular environmental homeostasis.Consequently, based on the previous study, we aim to further demonstrate the mechanism how Nestin regulates the intracellular homeostasis. This paper is composed of the following parts:Part one: Nestin is involved in maintaining intracellular membrane system stability.Part two: Mechanism of Nestin regulating intracellular ROS production by affecting mitochondria dynamics.Part three: Mechanism of Nestin regulating intracellular antioxidant system via protecting Nrf2 protein stability.Part 1:Nestin is involved in maintaining intracellular membrane system stability1.1 Objective Previously, we found that various proteins might interact with intermediate filament protein Nestin using Co IP-MS and super-resolution microscopy assays. Further analysis showed that intermediate filament protein Nestin might be associated with a variety of organelles with membranes. Therefore, this project intends to make a general preliminary inquiry on the biological function of Nestin and its impact on intracellular membrane system's morphology and function.1.2 Methods 1.2.1 Detect the relationship between Nestin and mitochondria. We detected whether Nestin could bind to mitochondria by immunofluorescence(IF) staining of COX IV and Nestin, and mitochondrial fractions extraction for Western blot. We also detected mitochondrial functions of NSC compared to that of Nestin knockdown NSC.1.2.2 Detect whether Nestin played a role in NSC proliferation and differentiation by Nestin knockdown. IF staining of Ki67 and Nestin, and clonal neurosphere-forming assays were used to evaluate cell proliferation. After inducement of NSC differentiation, IF staining of Tuj-1 and GFAP was used to identify neurons from astrocytes.1.2.3 Detect the relation between Nestin and SERCA2 by IF and Co-immunoprecipitation(Co-IP).1.2.4 Detect the relation between Nestin and endoplasmic reticulum(ER). We detected changes related to ER in the case of Nestin knockdown by ER staining and ER stress level detection.1.2.5 Detect the relation between Nestin and cell nucleus. We detected changes related to nucleus in the case of Nestin knockdown by Nuclear shape measurement and nuclear skeleton protein(Lamin A/C and Lamin B) expression detection.1.2.6 Detect the relation between Nestin and Lamin A/C. We detected whether Nestin could locate in cell nucleus by IF staining and nuclear fractions extraction for Western blot. We also detected the possibility that Nestin could bind to Lamin A/C by Co-IP.1.3 Results 1.3.1 Nestin interacts with mitochondria directly and knockdown of Nestin results in mitochondrial metabolism changes.1.3.2 Knockdown of Nestin impaired neural stem cell proliferating ability and enhanced differentiating capacity.1.3.3 Nestin is combined together with the ER protein SERCA2.1.3.4 Nestin knockdown will cause ER morphological changes and ER Stress increasing.1.3.5 Nestin knockdown will cause nucleus malformation rate increased and loose the nuclear membrane.1.3.6 Nestin can translocate into nucleus and combine with nuclear skeleton protein Lamin A/C.1.4 Conclusion 1.4.1 Nestin can combine with mitochondria and influence the metabolic function of mitochondria, and then regulate the proliferation and differentiation of neural stem cells.1.4.2 Nestin is combined with ER calcium pump SERCA2 and can influence ER morphology and function.1.4.3 Nestin can translocate into nucleus and combine with nuclear skeleton protein Lamin A/C to influence the membrane structure stability.Part 2: Mechanism of Nestin regulating intracellular ROS production by affecting mitochondria dynamics2.1 Objective Our previous studies have indicated that the intermediate filament protein Nestin can bind to the mitochondria and affect its function. Therefore, this project constructed gastrointestinal stromal tumor cell lines GIST-T1 and GIST-882 with stable Nestin knockdown and further studied the specific mechanism of how Nestin influenced mitochondrial morphology and function as well as the biological behavior of cancer cells.2.2 Methods 2.2.1 Detect whether Nestin knockdown influenced cellular ROS content. Through Nestin knockdown in GIST cell lines(GIST-T1 and GIST-882), we detected levels of total intracellular ROS, extracellular ROS derived from NADPH oxidase(Nox) family and mitochondrial ROS.2.2.2 Detect the relationship between Nestin and mitochondria. We detected morphology changes of mitochondria in the case of Nestin knockdown by Mito Tracker staining.2.2.3 Detect the relationship among Nestin, proteins related to mitochondrial fusion and fission, and mitochondria. To classify their relationship, we used Q-PCR, Western blot, Co-IP, IF, and image analysis.2.2.4 Detect whether Nestin or Drp1 influenced cancer proliferation and migration. We established two GIST cell lines, one cloud be induced to knockdown Nestin with doxycycline, and the other overexpressed Drp1. Both in vitro and in vivo experiments including wound-healing, matrigel invasion assay and cancer xenograft were used in this section.2.2.5 Further confirm that Nestin expression was related to cancer proliferation and migration by testing primary cancer tissues. Western blot and IHC were majorly used in this section.2.3 Results 2.3.1 After Nestin was interfered, ROS level decreased. Further testing shows that the decreased ROS is from mitochondria.2.3.2 Knockdown of Nestin resulted that shape of mitochondria changed from fragmentation to elongation.2.3.3 Nestin is combined with mitochondrial fission protein Drp1. Nestin knockdown resulted in decreased expression of Drp1, then mitochondrial dynamics was changed.2.3.4 Nestin knockdown leads to decrease in cancer cell proliferation and migration in vitro and tumorigenicity in vivo.2.3.5 Nestin expression is positively correlated cancer proliferation and migration ability in sample from GIST patients.2.4 Conclusion 2.4.1 Intermediate filament Nestin is involved in the regulation of intracellular ROS. Knockdown of Nestin leads to intracellular total ROS and mitochondrial ROS decreasing.2.4.2 Knockdown of Nestin results in the alteration of mitochondrial morphology from fragmentation to elongation.2.4.3 Nestin is combined with mitochondrial fission protein Drp1. Knockdown of Nestin leads to translocation of Drp1 from mitochondria to cytoplasm, indicating that Nestin regulates mitochondrial dynamics through recruiting Drp1 to mitochondria.2.4.4 Nestin is closely related to the biological behavior of gastrointestinal stromal tumor cells. Knockdown of Nestin resulted in decreased proliferation and migration in cancer cells both in vivo and in vitro.Part 3 Mechanism of Nestin regulating intracellular antioxidant system via protecting Nrf2 protein stability3.1 Objective It has been reported that knockdown of Nestin can accelerate H2O2-induced neural stem cells apoptosis. Over-expression of Nestin has a protective effect on apoptosis induced by H2O2 which suggests that antioxidant capacity of neural stem cells after Nestin knockdown decreased. It indicated that Nestin may regulate cellular antioxidant capacity directly. Therefore, this project constructed cancer cell lines A549 and H1299 with stable nestin knockdown and further studyed the specific molecular mechanism of how Nestin regulates intracellular antioxidant system.3.2 Methods 3.2.1 Detect the relationship between Nestin and sensitivity of oxidative stress. Lung cancer cell lines A549 and H1299 transfected with sh Nestin or NTC vectors were exposed to H2O2. We compared the cell survival rate and death rate between sh Nestin and NTC samples using Annexin V/PI staining or LIVE/DEAD? viability/cytotoxicity assays.3.2.2 To detect the change of antioxidant system after Nestin knockdown in lung cancer cell lines, we used Q-PCR, GSH detection assay, SOD activity detection assay, co-staining of Nestin and Nrf2 following image analysis3.2.3 To clarify the relationship between Nestin and Nrf2, we used western blot, Co-IP, treatment with protein synthesis inhibitor(CHX) and protein degradation inhibitor(MG132).3.2.4 Detect the relation among Nestin, Nrf2, and Keap1. To clarify their relationship, we used Co-IP, IF, and image analysis.3.3 Results 3.3.1 Analyzing the Annexin V/PI flow cytometry and confocal pictures using Live/Dead assay kit, we find that Nestin knockdown leads to increased sensitivity to oxidative stress in cancer cells, increasing cell death.3.3.2 The detection of cell antioxidant system shows that the knockdown of Nestin results in the elevated degradation of Nrf2 and decrease of the antioxidant capacity of cancer cells.3.3.3 Both in the normal state or under oxidative stress, the knockdown of Nestin can lead to a decrease of Nrf2 protein in the cancer cells, mainly through the ubiquitin- proteasome degradation pathway.3.3.4 Nestin can bind to Keap1 in a competitive way to reduce the degradation of Nrf2 levels.3.4 Conclusion 3.4.1 Nestin is related to the antioxidant capacity of cancer cells directly. Nestin knockdown can lead to increased sensitivity of cancer cells to oxidative stress, and increased mortality.3.4.2 Nestin knockdown leads to a decrease in antioxidant capacity of cancer cells, and an increase in Nrf2 degradation, which indicates that Nestin can regulate the antioxidant capacity of cancer cells by Nrf2.3.4.3 Nrf2 protein is protected by inhibiting binding with Keap1 and ubiquitin dependent degradation via Nestin competitively binding with Keap1, upregulating intracellular antioxidant system.3.4.4 Nestin knockdown leads to the elevated ubiquitination levels of Nrf2, which promotes degradation via ubiquitin-proteasome pathways in cancer cells. |
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