Investigating the inner world of stressed goblet cell

Human body consists of 37.2 trillion cells. Amongst them, the high protein turn over cells such as goblet, beta cells etc. do overwork to produce sufficient protein. Such cells are prone to high cell stress. Excessive cell stress eventually leads to triggering of cell death. Initiating anti-apoptotic signaling or triggering cell death depends to a great extent on the nature or source of cellular stress and cell type. Interplay between each stress response eventually determines the fate of stressed cell. Numerous factors induce cell death by a number of pathways including apoptosis, autophagy and necrosis. Not surprisingly, some of the pathways are interrelated to each other through a mediator that could articulate the entire mechanism. These stress responses are a hallmark of numerous diseases including neurodegenerative diseases, diabetes and cancer. Understanding the cross-talk between different intrinsic cell stress responses will help to develop new therapeutic targets and hence lead to the development of novel therapeutics. Genetic and environmental insults impede the functioning of cells to fold in the ER leading to building up of misfolded proteins leading to a condition called as endoplasmic reticular stress (ERS). Excessive ERS is a hallmark of several diseases including ulcerative colitis, cancer, diabetes and Parkinson's disease. Accumulating evidence suggests that the goblet cell-derived Mucin 2 (Muc2) is a major component of the immune system and that perturbations in Muc2 lead to an ulcerative colitis-like phenotype. The animal model of Winnie carries a missense mutation in Muc2 that lead to Muc2 accumulation in goblet cells. Although, colon pathology in the Winnie model has been extensively studied, Winnie goblet cells are characterized by ER stress, hyperproliferation and a striking absence of apoptosis leaving a huge question mark. This suggests that additional regulatory Muc2-associated mechanisms regulate the cellular stress response. On the other hand, sustained ERS is implicated in aggressive metastasis of cancer cells and increased tumor cell proliferation. Cancer cells activate the unfolded protein response (UPR), which aids in cellular survival and adaptation to harsh conditions. Inhibition of apoptosis, in contrast, is a mechanism adopted by cancer cells with the help of the inhibitor of an apoptosis (IAP) class of proteins such as Survivin to evade cell death and gain a proliferative advantage. In this present project there was also a scope for future cancer therapeutics where we have employed unconjugated bilirubin (UCB). Mildly elevated serum UCB concentrations are associated with protection against disease conditions underpinned by cellular and metabolic stress. My doctoral research thesis consists of six chapters. Chapter 1 is an overall introduction to the rational of the research, hypothesis and aims of the research project and a brief outline of ERS and cell death. Chapter 2 is a review article that joins various gaps in the literature regarding cell stress and summarizes the role of various components of apoptosis and inhibition of apoptosis. Chapter 3 details the proteomic approach for Winnie goblet cells to unravel the key players involved in evading programmed cell death and aiding in proliferation. The chapter also reveals the functional confirmation denoting the proteins involved. In chapter 4, I have further explored the molecular mechanisms correlating ERS and apoptosis which were unanswered in the 3rd chapter. The chapter covers the effect of ERS on ERS markers and inhibition of apoptosis protein, Survivin; inflammation; cancer cell apoptosis and proliferation. The chapter also deals with the converse effect of Survivin inhibition over ERS exhibiting downstream effects like ERS markers, inflammation and cell death. I have also elucidated the effects of ERS over cell invasion and migration. Chapter 5 involves therapeutics where I have incorporated unconjugated bilirubin (UCB) against ERS. The chapter covers the effect of UCB over inflammation induced by TUN, cancer cell apoptosis and proliferation. Finally, chapter 6 is a comprehensive discussion of all the results obtained from the research objectives and includes potential directions for future work arising from this research project. Methods employed in my study: Initially to elucidate the molecular mechanisms underlying ulcerative colitis, I have compared the proteomes of purified goblet cells from Winnie and wild type mice. Goblet cell proteins were analysed by SDS-PAGE and nano-LCMS. Proteins were identified using the search engine MaxQuant, compared for protein abundance and subjected to subsequent bio-informatics analysis. All the key proteins were functionally proved by immune fluorescence and RT-PCR. We have initially elucidated the lack of apoptosis in Winnie colon and also shown the characterization of goblet cells isolated. In Winnie mice, proteins with the following functional terms were found to be dysregulated: mitochondrial function, carbon metabolism, unfolded protein response, oxidative stress, amino acid metabolism, translation, cell structure and protein transport. In addition, multiple mitochondrial proteins with disturbed post-translational processing were identified. Subsequent gene expression and immunofluorescence analysis supported the proteomics results. In order to learn the association between ERS and apoptosis, I have initially verified the expression of Survivin (inhibitor of apoptosis) in Winnie (a mouse model of chronic ERS) colon tissues by using immunohistochemistry (IHC) and immunofluorescence (IF) in comparison with wild type Blk6 mice. Additionally, I isolated the goblet cells and determined the expression of Survivin by IF and protein validation. Tunicamycin was utilized at a concentration of 10 ˜í¬¿g/mL to induce ERS in the LS174T cell line and the gene expression of the ERS markers was measured. This was followed by determination of inflammatory cytokines. Inhibition of ERS was carried out by 4Phenyl Butyric acid (4PBA) at a concentration of 10 mM to assess whether there was a reciprocation effect. The downstream cell death assays including caspase 3/7, Annexin V, and poly (ADP-ribose) polymerase (PARP) cleavage were evaluated in the presence of ERS and absence of ERS, which was followed by a proliferative assay (EdU click) with and without ERS. Correspondingly, I have inhibited Survivin by YM155 at a concentration of 100 nM and observed the succeeding ERS markers and inflammatory markers. I also verified the caspase 3/7 assay to screen the intrinsic cell death pathway. I evaluated the positive effects of UC against ERS mediated effects like ERS and inflammatory markers like GRP78, NLRP3, IL-1b, XBP1, PERK and ATF6. I have also verified the effect of UCB against inflammation induced by TUN through bioplex of cytokines including IL-8, IL-10, IL-4 and TNF˜í¬±. Apoptosis which is one of the prime aspects of cancer therapeutics was also assessed by assays like Annexin V, PARP and caspase 3. I have also gauged the potential anti proliferative effects of UCB. Through my results, I have initially enumerated the lack of apoptosis in Winnie colon and also shown the characterization of goblet cells isolated. In Winnie mice, proteins with the following functional terms were found to be dysregulated: mitochondrial function, carbon metabolism, unfolded protein response, oxidative stress, amino acid metabolism, translation, cell structure and protein transport. In addition, multiple mitochondrial proteins with disturbed post-translational processing were identified with subsequent gene expression and IF analysis which supported the proteomics results. Secondly, I have demonstrated that ERS inhibition not only significantly reduced the UPR genes (Grp78, ATF6, PERK, and XBP1) along with Survivin but also downregulated the inflammatory markers such as IL8, IL4, and IL6, which suggests a positive correlation between ERS and the inhibition of apoptosis. Furthermore, I have provided evidence that ERS inhibition promoted apoptosis in LS174T cells and shortened the proliferation rate. Moreover, Survivin inhibition by YM155 led to a comparable effect as that of ERS inhibition, which includes attenuation of ERS genes and inflammatory markers as well as the promotion of programmed cell death via the caspase 3/7 pathway. To determine the potential therapeutic efficacy of UCB and establishing it as an anti ERS and anti-inflammatory agent, I have tested it in an in vitro model of gut inflammation.TUN (10 ˜í¬¿g/mL) was used to induce endoplasmic reticular stress (ERS) affecting N-glycosylation in LS174T cells. Cultured cells were investigated with addition of UCB at doses 0.1, 1 and 10 ˜í¬¿M (resulting in bilirubin:albumin ratios of 0.325‚ÄövÑv¨0.003) against ER stress-mediated effects including inflammation, cell survival (determined by apoptosis) and proliferation. Gene expression of ER stress markers (Grp78, Perk, XBP1 and ATF6) were evaluated in addition to cytokine concentrations in media after six hours of treatment. We then verified the potential role of UCB in executing programmed cell death via PARP, Caspase 3 and Annexin V assays and further explored cell proliferation using the Click-iT EdU assay. A dose of 10 ˜í¬¿M UCB most potently reduced TUN-mediated effects on enhanced UPR markers, inflammatory cytokines and proliferation; however all the doses (i.e.0.1‚ÄövÑv¨10 ˜í¬¿M) reduced the expression of ER stress and inflammatory markers Grp78, NLRP3, IL1-b, XBP1, PERK and ATF6. Furthermore, media concentrations of pro-inflammatory cytokines IL-8, IL-4 and TNF˜í¬± decreased and the anti-inflammatory cytokine IL-10 increased (p<0.05). A dose of 10 ˜í¬¿M UCB initiated intrinsic apoptosis via caspase 3 and in addition reduced cellular proliferation. Collectively, these data indicate that co treatment with UCB resulted in reducing ER stress response to TUN in gastrointestinal epithelial cells, reduc...