Dual functions of caspase 4 in apoptosis and inflammation

DUAL FUNCTIONS OF CASPASE-4 IN APOPTOSIS AND INFLAMMATION UMAYAL LAKSHMANAN INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 DUAL FUNCTIONS OF CASPASE-4 IN APOPTOSIS AND INFLAMMATION UMAYAL LAKSHMANAN M. Sc. (BIOTECHNOLOGY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor, Professor Alan Porter, for providing me the wonderful opportunity to pursue my PhD degree in his laboratory. I am especially thankful for his keen insight and perception in solving problems and remain grateful to Alan for his continuous encouragement, support, as well as guidance throughout these years. I am thankful to my graduate supervisory committee, Drs. Li BaoJie and Cao Xin Min for their constructive suggestions and critical comments. I would also like to thank past and present members of the AGP laboratory for their helpful discussion, technical assistance, cooperation, and friendship. My special thanks go to Dr. Alexander Godo Urbano for his collaboration during the initial stages of his AIF project and Dr. Li Lei for her special friendship, right from day one. I would like to express my heartfelt appreciation to my family for their understanding and unwavering support in every endeavor of mine. i TABLE OF CONTENTS ACKNOWLEDGEMENTS . i TABLE OF CONTENTS ii LIST OF FIGURES v LIST OF TABLES vii ABBREVIATIONS .viii LIST OF PUBLICATIONS x SUMMARY . xi 1. CHAPTER 1. Introduction 1.1 Caspases 1.1.1 Functional classification of caspases . 1.2 Caspase-4 1.2.1 History 1.2.2 Chromosomal position of casp-4 gene in the human genome . 1.2.3 Caspase-4 . 1.2.4 Possible functional murine othologue 1.3 Apoptosis 15 1.3.1 Types of apoptosis . 16 1.3.2 Endoplasmic reticulum and apoptosis . 19 1.4 Inflammation . 26 1.4.1 Innate immunity . 27 1.4.2 Toll like receptors (TLRs) . 28 1.4.3 LPS and TLR4 . 31 1.4.4 TLR4 signaling 33 1.5 Thesis rationale . 43 2. CHAPTER 2. Materials and Methods 45 2.1 Chemicals and reagents 45 2.2 Cell culture 46 2.3 Transfection of mammalian cells 47 2.3.1 Transient transfection using LIPOFECTIN . 47 2.3.2 Transient transfection using FUGENE6 47 2.3.2 Stable transfection by electroporation . 48 2.4 Molecular cloning . 49 2.4.1 Construction of expression plasmids . 49 2.4.2 Preparation of Escherichia coli competent cells 50 ii 2.4.3 DNA transformation 51 2.4.4 DNA preparation 51 2.5 Polymerase chain reaction (PCR) . 53 2.6 Site-directed mutagenesis . 54 2.7 Sytox/Hoechst (S/H) DNA staining 55 2.8 Cell death assay 55 2.9 Reporter assay . 57 2.10 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . 57 2.11 Western blot analysis 58 2.12 Phosphorylation detection . 58 2.13 RNA preparation . 59 2.14 Human cytokine array, RT-PCR and ELISA assays 61 2.14 Preparation of whole cell lysates 62 2.15 Preparation of nuclear extracts . 62 2.16 Co-immunoprecipitation . 62 2.17 Short hairpin RNA targeting of caspase-4 63 3. CHAPTER 3. Caspase-4 plays a role in ER stress-mediated apoptosis and polyglutamine aggregate-induced apoptosis . 66 3.1 Caspase-4 levels are massively reduced in shRNA knockdown stable clones. 66 3.2 Caspase-4 is not involved in the major extrinsic and intrinsic apoptotic pathways . 67 3.3 Caspase-4 is localized mainly to the endoplasmic reticulum . 69 3.4 ER stressors and apoptosis 70 3.5 Caspase-4 is processed in response to ER stress 72 3.6 Involvement of caspase-4 in ER stress-induced apoptosis . 75 3.7 ER stress induces G2/M arrest preferentially in the caspase-4-deficient SHEP1 cell population . 77 3.8 Involvement of caspase-4 in polyglutamine aggregate-induced ER stress . 82 3.9 Discussion . 85 4. CHAPTER 4. Caspase-4 in innate immunity: Caspase-4 interacts with TRAF6 and mediates LPS-induced NF-κB activation and IL-8 and MIP-1β production . 90 4.1 Stable knock down of caspase-4 in the human monocytic cells, THP1 . 90 iii 4.2 Defects in secreted cytokines in the caspase-4 knockdown clones 91 4.3 Reduced LPS-stimulated up-regulation of specific cytokine mRNAs in caspase4 knockdown cells 93 4.4 Caspase-4 mediates LPS-induced IL-1β production 98 4.5 Caspase-4 mediates LPS-induced cytokine induction through NF-κBdependent trans-activation 101 4.6 Caspase-4 interacts with endogenous and transfected TRAF6 . 106 4.7 Intact TRAF6-binding motif in caspase-4 important for TRAF6-caspase-4 interaction . 109 4.8 Reconstitution of caspase-4 in the knocked down clones restores the original phenotype 112 4.8 Discussion . 116 5. CHAPTER 5. Implications and future directions . 123 iv LIST OF FIGURES Figure 1.1 Caspase structure and activation. Figure 1.2 Caspase-4 predicted structure. Figure 1.3 Organization of casp-4 on human chromosome 11 . Figure 1.4 Chromosomal organization and phylogenetic relationship of the inflammatory group of caspases. . Figure 1.5 Caspase-activation pathways of apoptosis:. 17 Figure 1.6 ER stress and unfolded protein response (UPR). . 21 Figure 1.7 UPR and apoptosis. Post-adaptive phase of UPR. 24 Figure 1.8 TLR and ligands. 31 Figure 1.9: LPS structure. . 32 Figure 1.10 LPS signaling via TLR4. 34 Figure 1.11 TRAF6 binding site . 38 Figure 1.12 NF-κB canonical pathway . 40 Figure 3.1 Stable knock-down of caspase-4 in SHEP1 cells. . 66 Figure 3.2 No changes in the levels of apoptosis in caspase-4 knockdown cells with common apoptotic inducers. . . 68 Figure 3.3 Localization of caspase-4 in ER. . . 69 Figure 3.4 ER stress reagents induce cell death by apoptosis and up-regulate the ER stress marker GRP78. . 71 Figure 3.5 Caspase-4 is processed in response only to ER stress. . 74 Figure 3.6 Caspase-4 knockdown protects cells against ER stress-induced apoptosis. . . 76 Figure 3.7 Caspase-4 knockdown results in G2/M arrest following treatment with ER stressors. . 81 Figure 3.8: PolyQ(72) induces cell death by apoptosis. . . 82 Figure 3.9: Polyglutamine aggregates induce ER stress and caspase-4 processing. . 83 Figure 3.10 Caspase-4 is involved in polyglutamine aggregate-induced apoptosis. transfection. 84 Figure 4.1 Stable knockdown of caspase-4 in the human monocytic cell line, THP1 . 90 v Figure 4.2 Defects in secreted cytokines in the caspase-4 knockdown clones . 92 Figure 4.3 Reduced LPS-stimulated yields of specific cytokine mRNAs in caspase-4 knockdown cells. 94 Figure 4.4 Reduced LPS-stimulated up-regulation of specific cytokine mRNAs in caspase-4 knockdown cells. . 96 Figure 4.5 Synthesis of IL-1β mRNA and secretion of IL-1β are compromised in caspase-4 knockdown clones. . . 99 Figure 4.6. Reduced NF-κB activation and nuclear translocation of the p65 subunit of NF-κB in caspase-4 knockdown cells. . 100 Figure 4.7 Inhibited nuclear translocation of p65 subunit of NF-κB, reduced IκBα degradation and IKKβ phosphorylation, in caspase-4 knockdown cells. . . 102 Figure 4.8. NF-κB inhibitor blocks LPS-induced up-regulation and secretion of IL-8 and MIP-1β. . 105 Figure 4.9 LPS induces endogenous caspase-4 to interact transiently with endogenous TRAF6 and IRAK1. . 108 Figure 4.10 TBS of caspase-4. . 110 Figure 4.11 TRAF6-binding site in caspase-4 is essential for LPS-induced TRAF6caspase4 interaction . 112 Figure 4.12 Reconstitution of caspase-4 in the shRNA clones restores the NF-κB activity after LPS stimulation. . 113 Figure 4.13 Reconstitution of caspase-4 in the KD clones restores the chemokines’ synthesis . 114 Figure 4.14 Speculative model showing the position occupied by caspase-4 in LPS signaling. 117 Figure 5.1 ER stress response decision between survival and apoptosis. 125 Figure 5.2. Diagrammatic summary of signaling by misfolded proteins. 131 vi LIST OF TABLES Table 2.1 Antibodies used in the research 45 Table 2.2 Stable cell lines used in the current study . 48 Table 2.3 List of oligonucleotides used for caspase-4 knock-down. 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Circulation 111, 1814-1821. 147 [...]... stress-induced apoptosis and amyloid beta (Aβ)-induced apoptosis This was disputed (Obeng and Boise, 2005) in studies using cell lines lacking the expression of caspase- 4 and using a putative caspase- 4 fluorogenic substrate, LEVD-AFC It is possible that the role of caspase- 4 in ER stress-induced apoptosis is cell line specific (Hitomi et al., 20 04) Later on, the role of caspase- 4 in various types of ER... resulting in a caspase cascade that culminates in specific cleavage of cellular proteins 1.1.1 Functional classification of caspases Caspases can be functionally classified into two broad groups viz., apoptotic caspases and inflammatory caspases The mammalian and mouse apoptotic group comprises caspases 2, 6, 7, 8, 9 and 10, while the inflammatory group includes caspases 1, 4, and 5 in mammals and caspases... and MIP-1β secretion in THP1 cells, I investigated the role of caspase- 4 in NF-κB signaling LPS-induced NF-κB nuclear translocation and activation were inhibited in all caspase- 4- deficient clones LPS stimulated endogenous caspase- 4 and TRAF6 to interact; likewise, transfected caspase- 4 (but not caspase- 1) interacted with exogenous TRAF6 Mutation of a TRAF6-binding motif (PPESGE) that I identified in. .. mediated apoptosis Here again using DN caspase- 12 does not prove the point Similarly, apoptosis As the cell line is not expressing caspase- 12 in the first place, how is it possible to see any effect with the DN caspase- 12? Another glaring shortcoming is the lack of re-expression of caspase- 4 in the cell line lacking caspase- 4 The figures 2 and 3 in the paper (Obeng and Boise, 2005) shows that UPR was induced... caspase- 4/ 12 activity by using a fluorogenic caspase substrate LEVD-AFC in all their cell lines and argue that this activity is detected even in cell lines lacking caspase- 4 and -12 There is no known substrate of caspase- 4 and LEVD is not specific They use the same substrate for caspase- 12 lines, as caspase- 12 has 48 % homology to caspase- 4 There is not even a predicted site for caspase- 12 and using... same locus in humans but are absent in mice (Martinon and Tschopp, 20 04) , suggesting more complicated regulation in humans Caspase- 4 has the highest homology with murine caspase- 12 as well as caspase- 11 Sequence comparison of the caspase domain and prodomains of the inflammatory caspases suggests that both caspase- 4 and caspase- 5 probably arose from this mouse region of caspase- 11 through gene duplication... caspase- 4, I generated stable caspase- 4- deficient human THP1 monocytic cell lines, which exhibited substantially reduced LPS-induced secretion of several chemokines and cytokines, including IL-8, MIP-1β, MIP-3α and IL-1β The LPS-induced expression of the mRNAs encoding these cytokines was correspondingly reduced in the caspase4 -deficient clones Since a specific NF-κB inhibitor blocked LPS-induced IL-8 and. .. boxes interspersed by introns 6 1.2.3 Caspase- 4 For nearly a decade, nothing was known about caspase- 4 as it does not have an exact mouse homologue Caspase- 4 has 59% homology to caspase- 11 and 48 % homology to caspase- 12 In 20 04, caspase- 4 was shown to have a role in endoplasmic reticulum stress-mediated apoptosis (Hitomi et al., 20 04) , where caspase- 4 was shown to localize to the ER and participate in. .. becomes interesting to investigate whether caspase- 4 might have a role in ER stress-mediated apoptosis Caspase- 12 is also involved in hypo-responsiveness to LPS-induced production of cytokines like IL-1β, IL-8, etc (Saleh et al., 20 04) A SNP in human caspase- 12 leads to either a truncated protein containing the N-terminal CARD (caspase recruitment domain) or a full length variant that is enzymatically inactive... 20 04) Caspase- 12 in 80% of the human population is truncated as a result of a single nucleotide polymorphism (SNP) in the fourth exon, while only 20% of individuals of African descent express the full length variant (Scott and Saleh, 2007) which is involved in inflammation (Saleh et al., 20 04) The full length caspase- 12 is enzymatically inactive, instead acting as a decoy caspase that counters LPS-induced . DUAL FUNCTIONS OF CASPASE-4 IN APOPTOSIS AND INFLAMMATION UMAYAL LAKSHMANAN INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 DUAL FUNCTIONS. 3.8 Involvement of caspase-4 in polyglutamine aggregate-induced ER stress 82 3.9 Discussion 85 4. CHAPTER 4. Caspase-4 in innate immunity: Caspase-4 interacts with TRAF6 and mediates LPS-induced. 4.6 Caspase-4 interacts with endogenous and transfected TRAF6 106 4.7 Intact TRAF6-binding motif in caspase-4 important for TRAF6 -caspase-4 interaction 109 4.8 Reconstitution of caspase-4 in