Luận án: Đóng góp của Transporter Kẽm đến Tự thực và Lão hóa Mạch máu - Yitong Zhao, FSU

Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2018 Contribution of Zinc Transporters to Autophagy and Vascular Senescence Yitong Zhao Follow this and additional works at the DigiNole: FSU's Digital Repository. For more information, please contact lib-ir@fsu.edu FLORIDA STATE UNIVERSITY COLLEGE OF HUMAN SCIENCES CONTRIBUTION OF ZINC TRANSPORTERS TO AUTOPHAGY AND VASCULAR SENESCENCE By YITONG ZHAO A Dissertation submitted to the Department of Nutrition, Food and Exercise Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2018 Yitong Zhao defended this dissertation on June 14, 2018. The members of the supervisory committee were: Gloria Salazar Professor Directing Dissertation Cathy W. Levenson University Representative Bahram H.

Arjmandi Committee Member Shridhar K. Sathe Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements ii TABLE OF CONTENTS List of Figures. iv List of Tables. vii CHAPTER 1 INTRODUCTION.

1 CHAPTER 2 LITERATURE REVIEW. 5 CHAPTER 3 SPECIFIC AIMS AND HYPOTHESES. 23 CHAPTER 4 MATERIALS AND METHODS. 48 APPENDIX A FIGURES AND TABLES.

55 APPENDIX B IACUC ASSURANCE LETTER. 102 iii LIST OF FIGURES Figure 1. The signaling inducing cell cycle arrest. Types of senescence.

Effect of different minerals on autophagy. The effect of zinc on autophagy is dose-dependent. Number of autophagosomes is increased by zinc. p62 co-localizes with Golgi apparatus.

Zinc increases p62 protein levels without affecting its transcription. Zinc inhibits proteasome degradation pathway increasing p62 protein levels. Zinc transporter 2 (ZnT2), ZnT3 and ZnT10 increase p62 protein levels and LC3 II/I ratio. ZnT3 and ZnT10 interact with p62.

Zinc transported into endosomes by ZnT3 upregulates p62 expression. ZnT3 is required in zinc induced autophagy in MASMs. Number of autophagosomes is increased by zinc in ZnT3+/+, but not in ZnT3-/- MASMs. p62 is degraded through both lysosomal and proteasome degradation pathways in MASMs.

ZnT3 and ZnT10 regulate p62 protein levels. p62 is required in zinc-regulated autophagy. The increased senescence in ZnT3-/- MASMs is decreased by rapamycin. ZnT3-/- mice have higher senescence.

Senescence and p62 expression are increased in ZnT3-/- male mice. Senescence and p62 expression are increased in ZnT3-/- female mice. Senescence and plaque formation are increased in ApoE-/-ZnT3-/- male mice. Senescence is increased, and autophagy is inhibited in ApoE-/-ZnT3-/- male mice.

Atherosclerosis is not increased in ApoE-/-ZnT3-/- female mice. 77 v LIST OF TABLES Table 1. List of buffer preparation. The similarities and differences among senescence, apoptosis, and autophagy.

Quantification of markers in 4-month-old male mice. Quantification of markers in 4-month-old female mice. Quantification of markers in male mice fed LFD or HFD. 83 vi ABSTRACT Atherosclerosis is the leading cause of death among all age-related diseases.

One potential way to attenuate aging is to reduce the accumulation of aging cells, also called senescent cells. Our lab previously reported that zinc overload and deletion of zinc transporters contribute to senescence development in vascular smooth muscle cells (VSMCs). However, the detailed mechanism remains unclear. Autophagy, a protective mechanism removing dysfunctional organelles and proteins, has been reported to delay senescence development.

However, it is unknown whether the upregulation of autophagy by zinc and zinc transporters is required for the inhibition of senescence. Additionally, p62, an autophagy adaptor degraded through autophagy, has been reported to attenuate senescence in mice. However, it remains unknown whether p62 protein levels are regulated by zinc or zinc transporters. In this dissertation, we found that in HEK293 cells, zinc stimulated autophagy and inhibited proteasome degradation.

However, p62 level was increased by zinc. Zinc changed the location of p62 and protected p62 from degradation. Similarly, overexpression of zinc transporters (ZnT), especially ZnT3, stimulated autophagy and increased the p62 level. Additionally, ZnT3 interacted with p62 and protected p62 from degradation pathways.

In VSMCs isolated from aortas of mice (MASMs), zinc and overexpression of ZnT3 also upregulated autophagy. Lack of ZnT3 or p62 in MASMs contributed to defective autophagy and increased senescence. Similarly, ZnT3-/- mice presented a higher senescence level and an increased level of p62, suggesting that autophagy is downregulated in ZnT3-/- mice. Male ApoE-/-ZnT3-/- mice fed with high fat diet (HFD) had more plaque suggesting ZnT3 is important in atherosclerosis in male.

However, atherosclerosis level was not affected by HFD in female ApoE-/-ZnT3-/- mice. Overall, in this dissertation, we uncovered a new role of ZnT3 as a critical regulator of autophagy, vascular senescence, and atherosclerosis. vii CHAPTER 1 INTRODUCTION Aging is one of the major risk factors in the development of age-related diseases like cardiovascular disease, which is the leading cause of death in the US (1). Aging is characterized by the accumulation of aged cells, which are also called senescent cells (2).

Senescent cells are characterized by permanent cell cycle arrest and the secretion of inflammatory molecules and cytokines like interleukins to the surrounding environment. This process is called senescence- associated secretory phenotype (SASP), which stimulates neighboring cells as senescence stimuli (3). Neighboring cells receive the stimuli and become senescent. In addition to these functional changes, senescent cells also present morphologic changes like enlargement and flattening (2).

Understanding the molecular mechanism of senescence development would help to design strategies to delay aging as well as age-related diseases. Cellular senescence is a complex process induced by multiple factors including telomere shortening, radiation and the excessive production of reactive oxygen species (ROS), which cause mutations of the DNA (1). There are two types of senescence, replicative senescence (4) and stress- induced premature senescence (SIPS). Replicative senescence refers to the limited proliferation capacity due to telomere shortening.

This type of senescence develops slowly because the telomere becomes shorter in each cell division. When the telomere is critically short and cannot support DNA replication, cell proliferation is blocked (5). On the other hand, SIPS is induced by stress conditions like the excess level of ROS and radiation. These stimuli cause DNA damage and lead to cell cycle arrest (6).

Excess levels of ROS can be caused by increased production from several sources, like NADPH oxidases (Nox) and dysfunctional mitochondria (7,8). Additionally, excess level of ROS could be also caused by defects in processes like autophagy, by which dysfunctional mitochondria are degraded (8). Autophagy is a catabolic process degrading dysfunctional organelles as well as abnormal proteins (9). Enhanced autophagy could reduce ROS level in cells and delay senescence development (10).

Autophagy is mediated by the formation of autophagosomes, which is regulated in part by autophagy-related proteins (ATG). The autophagosome contains cargo to be degraded as well as autophagy adaptor proteins, like sequestosome (SQSTM1), also called p62 (11). The formation of autophagosomes is indicated by the conversion of microtubule-associated protein 1 1A/1B-light chain 3-I (LC3-I) to LC3-II. LC3-II is the lipidated form of LC3-I that attaches to the membrane of the autophagosome and simulates its elongation and closure.

The autophagosome then fuses with lysosome forming an autolysosome, in which autophagy genes like LC3-II and p62 are degraded. Thus, successful autophagy is indicated by the degradation of p62, and the increased conversion from LC3-I to LC3-II (12). This protective process could be activated by oxidative stress and is downregulated during aging, leading to the accumulation of protein aggregates and dysfunctional mitochondria (13). During aging, cells produce excess level of ROS and accumulate abnormal proteins which are proposed to activate autophagy.

However, the continuous exposure to ROS and accumulation of abnormal proteins cause exhaustion of autophagy and eventually leads to the suppression of autophagy (14). Recent studies suggest that both excessive and defective autophagy could induce senescence (15,16). Thus, understanding the cellular mechanism regulating autophagy would help to delay the development of senescence. There are several factors that could affect the level of autophagy in addition to aging, like changes in zinc level and distribution (17).

Zinc homeostasis is altered during aging because of decreased intestinal absorption in elderly and defective regulation of zinc distribution in cells (18). In general, the concentration of free zinc in the cytosol is very low (10-9 M to 10-12 M) (19). Under the stimuli like ROS, zinc is released as free zinc from zinc-binding proteins like metallothioneins. The excess level of free zinc stimulates the expression of zinc-binding proteins to chelate the free zinc and upregulates autophagy, which protects the cell (20).

However, excess free zinc could also induce autophagic cell death (21,22). Lee and colleagues suggested that the exposure of astrocytes to free zinc (300 µM) for 10 min increased the amount of autophagosomes, indicating stimulated autophagy (23). However, this study did not assess whether the excess level of free zinc blocks the degradation of autophagosome content, which would also result in the accumulation of autophagosomes. Additionally, H2O2 exposure induces the release of free zinc to the cytosol in astrocytes.

Free zinc enters lysosome and triggers lysosomal membrane permeabilization (LMP) causing cell death (24). This study suggests that the amount and the distribution of zinc in the cell are important in the regulation of autophagy. The distribution of zinc, as well as the levels of free zinc, are tightly regulated by zinc-binding proteins and zinc transporters. Zinc-binding proteins, as mentioned above, store zinc in normal condition and protect cells through binding zinc when excess free zinc is induced by stimuli like ROS (25).

On the other hand, two families of zinc transporters transport 2 zinc in and out of the cytosol (26). The ZnT or SLC30A family transports zinc outside of cells or inside organelles to reduce the level of zinc in the cytosol. In contrast, the Zrt It-like protein (ZIP, solute carrier family 39A or SLC39A) family transports zinc into the cytosol (27). Among all zinc transporters, expression of ZnT3 and ZnT10 are altered during aging and age-related diseases like Alzheimer’s disease (AD).

The expression of ZnT3 decreases during aging and results in zinc deficiency in synaptic vesicles, which accelerates brain aging in a senescence-accelerated mice model (28). Brain samples, as well as frontal cortex samples from AD model, show a decreased expression of both ZnT3 and ZnT10 (29). Additionally, our lab previously reported that downregulation of these transporters enhances ROS level and induces senescence, while overexpression of ZnT3 and ZnT10 reduces ROS level and delays senescence in VSMCs in response to angiotensin II (Ang II) (30). Overexpression of ZnT3 and ZnT10 decreases cytosolic zinc and, as mentioned before, the altered distribution of zinc should affect autophagy.

However, it is unknown whether ZnT3 and ZnT10 participate in autophagy and whether zinc- and/or ZnT-regulated autophagy contribute to senescence. Very few studies investigated the role of zinc transporters in the regulation of cell fate. For instance, downregulation of ZnT4 inhibits lysosomal degradation, resulting in apoptosis in HeLa cells (31). Overexpression of ZnT2 accumulates zinc in mitochondria and lysosomes leading to lysosomal-mediated cell death in mammary epithelial cells (32).

Since lysosomes fuse with autophagosomes, it is likely that these zinc transporters should also have a role in autophagy. So far, no study showed the role of ZnT3 or ZnT10 in the regulation of autophagy, and whether ZnT-regulated autophagy could delay senescence. Thus, we investigated the role of ZnT3 and ZnT10 in the regulation of autophagy and the role of zinc-regulated autophagy in senescence and atherosclerosis development. In this dissertation, we found that in HEK293 cells zinc increases protein stability of p62 and increases the formation of autophagosomes.

We found that p62 is degraded through both proteasome and lysosomal degradation pathways and zinc inhibits the proteasome degradation pathway. Overexpression of ZnT3 and ZnT10 increases LC3 II/I ratio, suggesting autophagy is upregulated. Additionally, ZnT3 and ZnT10 both interact with p62, suggesting these zinc transporters re-localize p62 out of degradation pathways. To investigate whether zinc transporters are important in zinc-upregulated autophagy, we isolated VSMCs from ZnT3+/+ and ZnT3-/- mice aortas (MASMs).

Zinc increased p62 protein levels in a dose-dependent manner in ZnT3+/+ MASMs, but not in ZnT3-/- cells, indicating that ZnT3 is required to increase p62 protein levels by 3 zinc.