The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

Cheung, Siu Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 178-200). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vii / Chapter Chapter One --- General Introduction --- p.1 / Chapter 1.1 --- S100 Proteins --- p.1 / Chapter 1.1.1 --- Structure of S100 proteins --- p.2 / Chapter 1.1.2 --- Possible functions of S100 proteins --- p.4 / Chapter 1.1.3 --- Genomic organization of S100 genes --- p.6 / Chapter 1.1.4 --- Clinical importance of S100 proteins --- p.7 / Chapter 1.2 --- S100A1 Protein --- p.8 / Chapter 1.2.1 --- Possible functions of the S100A1 protein --- p.10 / Chapter 1.2.1.1 --- Regulation of cardiac and skeletal muscle contractility --- p.10 / Chapter 1.2.1.2 --- Functional roles in the central nervous system (CNS) --- p.12 / Chapter 1.2.1.3 --- Other possible functions of the S100A1 protein --- p.13 / Chapter 1.2.2 --- S100A1 knockout mice --- p.14 / Chapter 1.2.3 --- Relationships between S100A1 and S100B proteins --- p.16 / Chapter 1.3 --- S100B Protein --- p.18 / Chapter 1.3.1 --- Possible functions of S100B protein --- p.19 / Chapter 1.3.2 --- S100B knockout mice --- p.20 / Chapter 1.4 --- RNA interference --- p.22 / Chapter 1.4.1 --- Mechanisms of RNA interference --- p.24 / Chapter 1.4.2 --- Efficacy and selectivity of siRNA --- p.25 / Chapter 1.4.3 --- siRNA delivery --- p.27 / Chapter 1.5 --- Objective --- p.31 / Figures and legends --- p.34 / Chapter Chapter Two --- S100A1 expression in normal mouse embryos and characterization of S100A1 knockout mouse embryos --- p.40 / Chapter 2.1 --- Introduction --- p.40 / Chapter 2.2 --- Materials and Methods --- p.44 / Chapter 2.2.1 --- Mouse strains --- p.44 / Chapter 2.2.2 --- RNA extraction --- p.46 / Chapter 2.2.3 --- Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.46 / Chapter 2.2.4 --- Protein extraction --- p.48 / Chapter 2.2.5 --- Western blotting --- p.49 / Chapter 2.2.6 --- Immunohistochemical staining --- p.50 / Chapter 2.3 --- Results --- p.53 / Chapter 2.3.1 --- S100A1 mRNA expression in normal mouse embryo --- p.53 / Chapter 2.3.2 --- S100A1 protein expression in normal mouse embryos --- p.55 / Chapter 2.3.2.1 --- Temporal expression of the S100A1 protein --- p.55 / Chapter 2.3.2.2 --- Spatial expression of the S100A1 protein --- p.57 / Chapter 2.3.3 --- Morphological and histological characterization of SI00A1 knockout mouse embryos --- p.60 / Chapter 2.3.4 --- S100B protein expression pattern in Wt and S100A1 KO mouse embryos --- p.62 / Chapter 2.4 --- Discussion --- p.64 / Tables --- p.73 / Figures and legends --- p.76 / Chapter Chapter Three --- Knockdown of S100A1 in S100B in knockout mouse embryos --- p.118 / Chapter 3.1 --- Introduction --- p.118 / Chapter 3.2 --- Materials and Methods --- p.128 / Chapter 3.2.1 --- Mouse strains --- p.128 / Chapter 3.2.2 --- Short-interfering RNA (siRNA) --- p.129 / Chapter 3.2.3 --- In-uterus surgery --- p.130 / Chapter 3.2.4 --- RNA extraction and RT-PCR --- p.132 / Chapter 3.2.5 --- Immunohistochemical staining of S100A1 and S100B --- p.132 / Chapter 3.3 --- Results --- p.133 / Chapter 3.3.1 --- Characterization of S100B knockout mouse embryos --- p.133 / Chapter 3.3.2 --- S100A1 knockdown in S100B wild-type (Wt) mouse embryos --- p.133 / Chapter 3.3.3 --- S100A1 knockdown in S100B knockout (KO) mouse embryos --- p.139 / Chapter 3.4 --- Discussion --- p.146 / Tables --- p.153 / Figures and legends --- p.154 / Chapter Chapter Four --- General Discussion and Conclusions --- p.175 / Reference --- p.178

Diss. (sammanfattning) Stockholm : Karol. inst., 2007. / Härtill 4 uppsatser.

Proteins perform an incredible array of functions facilitated by a diverse set of biochemical properties. Changing these properties is an essential molecular mechanism of evolutionary change, with major questions in protein evolution surrounding this topic. How do new functional biochemical features evolve? How do proteins change following gene duplication events? I used the S100 protein family as a model to probe these aspects of protein evolution. The S100s are signaling proteins that play a diverse range of biological roles binding Calcium ions, transition metal ions, and other proteins. Calcium drives a conformational change allowing S100s to bind to diverse peptide regions of target proteins. I used a phylogenetic approach to understand the evolution of these diverse biochemical features. Chapter I comprises an introduction to the disseration. Chapter II is a co-authored literature review assessing available evidence for global trends in protein evolution. Chapter III describes mapping of transition metal binding onto a maximum likelihood S100 phylogeny. Transition metal binding sites and metal-driven structural changes are a conserved, ancestral features of the S100s. However, they are highly labile at the amino acid level. Chapter IV further characterizes the biophysics of metal binding in the S100A5 lineage, revealing that the oft–cited Ca2+/Cu2+ antagonism of S100A5 is likely due to an experimental artifact of previous studies. Chapter V uses the S100 family to investigate the evolution of binding specificity. Binding specificity for a small set of peptides in the duplicate S100A5 and S100A6 clades. Ancestral sequence reconstruction reveals a pattern of clade-level conservation and apparent subfunctionalization along both lineages. In chapter VI, peptide phage display, deep-sequencing, and machine-learning are combined to quantitatively reconstruct the evolution of specificity in S100A5 and S100A6. S100A5 has subfunctionalized from the ancestor, while S100A6 specificity has shifted. The importance of unbiased approaches to measure specificity are discussed. This work highlights the lability of conserved functions at the biochemical level, and measures changes in specificity following gene duplication. Chapter VII summarizes the results of the dissertation, considers the implications of these results, and discusses limitations and future directions. This dissertation includes both previously published/unpublished and co- authored material.

Introduction: Recent findings and better understanding to the pathogenesis of rheumatic diseases contributed to the development of biological therapies targeting cytokines and immune cells. Several S100 proteins exert cytokine-like effects and participate in the regulation of the inflammatory process. The aim of this work was to study the role of selected S100 proteins in the activity and in the pathogenesis of the rheumatic diseases. Results: Our data show for the first time an association of S100A4 proteinwith RA disease activity and decrease of the bioactive form, but not the total amount of S100A4, after aplication of tumour necrosis factor (TNF) blocking biologic therapy in patients with RA. We demonstrated that in vitro S100A4 acts as a potent pro-inflammatory mediator inducing production of TNFα, interleukin (IL)-1β and IL-6 in PBMCs via Toll-like receptor 4 (TLR-4), transcription factor NFκB and tyrosine kinases erk1/2 and p38. Moreover, S100A4 can play an important role in the pathogenesis of inflammatory myopathies. S100A4 is present in the inflammatory infiltrate of the affected muscles and in the regenerating muscles and may act as a cytokine-like factor indirectly promoting muscle fiber damage by stimulating mononuclear cells to increase the synthesis of pro-inflammatory cytokines. We...

Rheumatic diseases are common, usually chronic, painful and to some extent invalidating medical conditions. Understanding of the disease pathogenesis is still very fragmentary. Hyperreactivity of the immune system and defect of autotolerance are probably contributed by local factors, which helps to explain, why some joints/muscles are more affected than others. All this results from a complex net of interactions between immune cells, synovial fibroblasts, chondrocytes, osteocytes, myocytes and other cells. In the submitted PhD thesis I have focused on three groups of molecules: regulatory RNAs, S100 proteins and autoantibodies. In the theoretical part, I sum up the current knowledge on their biogenesis, function and the role in rheumatology. In the investigative part, I present six original publications and one review on the role of those molecules in development of rheumatoid arthritis (RA) and idiopathic inflammatory myositis (IIM). One of the main studies was focused on expression of PIWI-interacting RNAs (piRNAs) in RA synovial fibroblasts (SF). piRNAs are small regulatory RNAs which in complex with PIWIL proteins regulate gene expression and silence transpozoms. piRNA expression was considered to be limited to germline and cancer cells. We have found 267 PIWI-interacting RNAs to be expressed...

Rheumatic diseases are common, usually chronic, painful and to some extent invalidating medical conditions. Understanding of the disease pathogenesis is still very fragmentary. Hyperreactivity of the immune system and defect of autotolerance are probably contributed by local factors, which helps to explain, why some joints/muscles are more affected than others. All this results from a complex net of interactions between immune cells, synovial fibroblasts, chondrocytes, osteocytes, myocytes and other cells. In the submitted PhD thesis I have focused on three groups of molecules: regulatory RNAs, S100 proteins and autoantibodies. In the theoretical part, I sum up the current knowledge on their biogenesis, function and the role in rheumatology. In the investigative part, I present six original publications and one review on the role of those molecules in development of rheumatoid arthritis (RA) and idiopathic inflammatory myositis (IIM). One of the main studies was focused on expression of PIWI-interacting RNAs (piRNAs) in RA synovial fibroblasts (SF). piRNAs are small regulatory RNAs which in complex with PIWIL proteins regulate gene expression and silence transpozoms. piRNA expression was considered to be limited to germline and cancer cells. We have found 267 PIWI-interacting RNAs to be expressed...

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第20292号 / 人健博第40号 / 新制||人健||4(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 岡 昌吾, 教授 藤井 康友, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

As ações da atividade física na neuroquímica dos neurônios, com enfoque às proteínas ligantes de cálcio (Ca2+), e o estado de ativação de células gliais da medula espinal do rato foram investigadas em preparados imuno-histoquímicos através da análise morfométrica e microdensitométrica com auxílio do computador. Ratos machos adultos foram divididos em dois grupos: treinado, cujos animais foram expostos à roda de corrida onde realizava atividade física espontânea, por um período de 4 e 14 noites; e sedentário, onde os animais foram mantidos em caixas individualizadas, sem a roda de corrida. Após os períodos determinados, os animais sofreram eutanásia e suas medulas espinais foram processadas para imunohistoquímica. Os ligantes de Ca2+ neuronal e glial foram avaliados pela imunorreatividade das proteínas calbindina e parvalbumina e, ainda, pela imunorreatividade da proteína S100 astrocitária. A atividade física voluntária promoveu uma diminuição na imunorreatividade da proteína calbindina em nível torácico no corno posterior (lâminas I e II de Rexed), assim como no núcleo espinal lateral após 14 dias. No nível lombar, também se observou uma diminuição da imunorreatividade no corno posterior (lâminas I e II de Rexed). Contudo os animais submetidos à atividade física voluntária por 4 dias apresentaram um aumento na área imunorreativa da proteína parvalbumina em relação ao seu controle. Efeito semelhante ocorreu no núcleo dorsal nos grupos que treinaram por 4 e 14 dias. Entretanto, no fascículo cuneiforme ocorreu uma diminuição da imunorreatividade à parvalbumina. Já em relação à proteína S100, os animais treinados apresentaram um aumento na imunorreatividade (spMGV) no corno anterior. Assim, conclui-se que a atividade física voluntária modificou a imunorreatividade das proteínas ligantes de Ca2+ na medula espinal, o que pode estar associado aos mecanismos de ativação intracelular realizados pelo cálcio, bem como a liberação de neurotransmissores na fenda sináptica. / Actions of the physical activity in the neurochemistry focuzing calcium-bindin proteins and the activation of the glial cells in the spinal cord of the rat were investigated with imunohistochemistry over. Male wistar adult rats were divided in two groups: trained, which animals exercised in the wheel running for 4 and 14 nigths; and sedentary, which animals were maintained in private box without wheel running. After that period rats were sacrificed and their spinal cords were processed to imunohistochemistry. Calcium-bindin proteins neuronal (parvalbumin and calbindin) and glial (S100) were evaluted. The activity promoted a decrese in the imunoreativite of the calbindin protein in the torácic level of the posterior horn (lamina I and II of Rexed), and lateral spine nucle after 14 days. In the lombar level, decrese in the posterior horn was also found. Animals submited to physic activity for 4 days showed an increased in the imunoreatived area of parvalbumin. Similar effect was observed all of groups that were treineds for 4 e 14 days. However, in the cuneiforne fascicule, parvalbumin decreased. The S100 protein showed decresed in the anterior horn. In conclusion volunteer phisical activity changed the pattern of the calcium-bindin protein immunoreactivity in the spinal cord, effect than can be associated to neuroplasticity.