Role and regulatory mechanisms of heat shock factor 2

Protein homeostasis is essential for cells to prosper and survive. Various forms of stress, such as elevated temperatures, oxidative stress, heavy metals or bacterial infections cause protein damage, which might lead to improper folding and formation of toxic protein aggregates. Protein aggregation is associated with serious pathological conditions such as Alzheimer’s and Huntington’s disease. The heat shock response is a defense mechanism that protects the cell against protein-damaging stress. Its ancient origin and high conservation among eukaryotes suggest that the response is crucial for survival. The main regulator of the heat shock response is the transcription factor heat shock factor 1 (HSF1), which induces transcription of genes encoding protective molecular chaperones. In vertebrates, a family of four HSFs exists (HSF1-4), with versatile functions not only in coping with acute stress, but also in development, longevity and cancer. Thus, knowledge of the HSFs will aid in our understanding on how cells survive suboptimal circumstances, but will also provide insights into normal physiological processes as well as diseaseassociated conditions. In this study, the function and regulation of HSF2 have been investigated. Earlier gene inactivation experiments in mice have revealed roles for HSF2 in development, particularly in corticogenesis and spermatogenesis. Here, we demonstrate that HSF2 holds a role also in the heat shock response and influences stress-induced expression of heat shock proteins. Intriguingly, DNA-binding activity of HSF2 upon stress was dependent on the presence of intact HSF1, suggesting functional interplay between HSF1 and HSF2. The underlying mechanism for this phenomenon could be configuration of heterotrimers between the two factors, a possibility that was experimentally verified. By changing the levels of HSF2, the expression of HSF1-HSF2 heterotrimer target genes was altered, implementing HSF2 as a modulator of HSF-mediated transcription. The results further indicate that HSF2 activity is dependent on its concentration, which led us to ask the question of how accurate HSF2 levels are achieved. Using mouse spermatogenesis as a model system, HSF2 was found to be under direct control of miR-18, a miRNA belonging to the miR-17~92 cluster/Oncomir-1 and whose physiological function had remained unclear. Investigations on spermatogenesis are severely hampered by the lack of cell systems that would mimic the complex differentiation processes that constitute male germ cell development. Therefore, to verify that HSF2 is regulated by miR-18 in spermatogenesis, a novel method named T-GIST (Transfection of Germ cells in Intact Seminiferous Tubules) was developed. Employing this method, the functional consequences of miR-18-mediated regulation in vivo were demonstrated; inhibition of miR- 18 led to increased expression of HSF2 and altered the expression of HSF2 target genes Ssty2 and Speer4a. Consequently, the results link miR-18 to HSF2-mediated processes such as germ cell maturation and quality control and provide miR-18 with a physiological role in gene expression during spermatogenesis.Taken together, this study presents compelling evidence that HSF2 is a transcriptional regulator in the heat shock response and establishes the concept of physical interplay between HSF2 and HSF1 and functional consequences thereof. This is also the first study describing miRNA-mediated regulation of an HSF.

Factors Regulating Chondrogenic Differentiation

Chondrogenesis is a co-ordinated differentiation process in which mesenchymal cells condensate, differentiate into chondrocytes and begin to secrete molecules that form the extracellular matrix. It is regulated in a spatio-temporal manner by cellular interactions and growth and differentiation factors that modulate cellular signalling pathways and transcription of specific genes. Moreover, post-transcriptional regulation by microRNAs (miRNAs) has appeared to play a central role in diverse biological processes, but their role in skeletal development is not fully understood. Mesenchymal stromal cells (MSCs) are multipotent cells present in a variety of adult tissues, including bone marrow and adipose tissue. They can be isolated, expanded and, under defined conditions, induced to differentiate into multiple cell lineages including chondrocytes, osteoblasts and adipocytes in vitro and in vivo. Owing to their intrinsic capability to self-renew and differentiate into functional cell types, MSCs provide a promising source for cell-based therapeutic strategies for various degenerative diseases, such as osteoarthritis (OA). Due to the potential therapeutic applications, it is of importance to better understand the MSC biology and the regulatory mechanisms of their differentiation. In this study, an in vitro assay for chondrogenic differentiation of mouse MSCs (mMSCs) was developed for the screening of various factors for their chondrogenic potential. Conditions were optimized for pellet cultures by inducing mMSC with different bone morphogenetic proteins (BMPs) that were selected based on their known chondrogenic relevance. Characterization of the surface epitope profile, differentiation capacity and molecular signature of mMSCs illustrated the importance of cell population composition and the interaction between different populations in the cell fate determination and differentiation of MSCs. Regulation of Wnt signalling activity by Wnt antagonist sFRP-1 was elucidated as a potential modulator of lineage commitment. Delta-like 1 (dlk1), a factor regulating adipogenesis and osteogenesis, was shown to exhibit stage-specific expression during embryonic chondrogenesis and identified as a novel regulator of chondrogenesis, possibly through mediating the effect of TGF-beta1. Moreover, miRNA profiling demonstrated that MSCs differentiating into a certain lineage exhibit a specific miRNA expression profile. The complex regulatory network between miRNAs and transcription factors is suggested to play a crucial role in fine-tuning the differentiation of MSCs. These results demonstrate that commitment of mesenchymal stromal cells and further differentiation into specific lineages is regulated by interactions between MSCs, various growth and transcription factors, and miRNA-mediated translational repression of lineage-specific genes.

The Cellular Oxygen Sensor PHD2 in Cancer Growth

Adequate supply of oxygen is essential for the survival of multicellular organisms. However, in several conditions the supply of oxygen can be disturbed and the tissue oxygenation is compromised. This condition is termed hypoxia. Oxygen homeostasis is maintained by the regulation of both the use and delivery of oxygen through complex, sensitive and cell-type specific transcriptional responses to hypoxia. This is mainly achieved by one master regulator, a transcription factor called hypoxiainducible factor 1 (HIF-1). The amount of HIF-1 is under tight oxygen-dependent control by a family of oxygen-dependent prolyl hydroxylase domain proteins (PHDs) that function as the cellular oxygen sensors. Three family members (PHD1-3) are known to regulate HIF of which the PHD2 isoform is thought to be the main regulator of HIF-1. The supply of oxygen can be disturbed in pathophysiological conditions, such as ischemic disorders and cancer. Cancer cells in the hypoxic parts of the tumors exploit the ability of HIF-1 to turn on the mechanisms for their survival, resistance to treatment, and escape from the oxygen- and nutrient-deprived environment. In this study, the expression and regulation of PHD2 were studied in normal and cancerous tissues, and its significance in tumor growth. The results show that the expression of PHD2 is induced in hypoxic cells. It is overexpressed in head and neck squamous cell carcinomas and colon adenocarcinomas. Although PHD2 normally resides in the cytoplasm, nuclear translocation of PHD2 was also seen in a subset of tumor cells. Together with the overexpression, the nuclear localization correlated with the aggressiveness of the tumors. The nuclear localization of PHD2 caused an increase in the anchorage-independent growth of cancer cells. This study provides information on the role of PHD2, the main regulator of HIF expression, in cancer progression. This knowledge may prove to be valuable in targeting the HIF pathway in cancer treatment.

Transcriptional Profiling of Organ‐Specific Autoimmunity in Human

Our understanding of the pathogenesis of organ‐specific autoinflammation has been restricted by limited access to the target organs. Peripheral blood, however, as a preferred transportation route for immune cells, provides a window to assess the entire immune system throughout the body. Transcriptional profiling with RNA stabilizing blood collection tubes reflects in vivo expression profiles at the time the blood is drawn, allowing detection of the disease activity in different samples or within the same sample over time. The main objective of this Ph.D. study was to apply gene‐expression microarrays in the characterization of peripheral blood transcriptional profiles in patients with autoimmune diseases. To achieve this goal a custom cDNA microarray targeted for gene‐expression profiling of human immune system was designed and produced. Sample collection and preparation was then optimized to allow gene‐expression profiling from whole‐blood samples. To overcome challenges resulting from minute amounts of sample material, RNA amplification was successfully applied to study pregnancy related immunosuppression in patients with multiple sclerosis (MS). Furthermore, similar sample preparation was applied to characterize longitudinal genome‐wide expression profiles in children with type 1 diabetes (T1D) associated autoantibodies and eventually clinical T1D. Blood transcriptome analyses, using both the ImmunoChip cDNA microarray with targeted probe selection and genome‐wide Affymetrix U133 Plus 2.0 oligonucleotide array, enabled monitoring of autoimmune activity. Novel disease related genes and general autoimmune signatures were identified. Notably, down‐regulation of the HLA class Ib molecules in peripheral blood was associated with disease activity in both MS and T1D. Taken together, these studies demonstrate the potential of peripheral blood transcriptional profiling in biomedical research and diagnostics. Imbalances in peripheral blood transcriptional activity may reveal dynamic changes that are relevant for the disease but might be completely missed in conventional cross‐sectional studies.

JNK, a versatile regulator of kinesin-1 transport and cytoskeleton dynamics

C-Jun N-terminal kinase (JNK) is traditionally recognized as a crucial factor in stress response and inducer of apoptosis upon various stimulations. Three isoforms build the JNK subfamily of MAPK; generally expressed JNK1 and JNK2 and brain specific JNK3. Degenerative potency placed JNK in the spotlight as potential pharmacological option for intervention. Unfortunately, adverse effects of potential drugs and observation that expression of only JNK2 and JNK3 are induced upon stress, restrained initial enthusiasm. Notably, JNK1 demonstrated atypical high constitutive activity in neurons that is not responsive to cellular stresses and indicated existence of physiological activity. This thesis aimed at revealing the physiological functions of JNK1 in actin homeostasis through novel effector MARCKS-Like 1 (MARCKSL1) protein, neuronal trafficking mediated by major kinesin-1 motor protein and microtubule (MT) dynamics via STMN2/SCG10. The screen for novel physiological JNK substrates revealed specific phosphorylation of C-terminal end of MARCKSL1 at S120, T148 and T183 both ex vivo and in vitro. By utilizing site-specific mutagenesis, various actin dynamics and migrations assays we were able to demonstrate that JNK1 phosphorylation specifically facilitates F-actin bundling and thus filament stabilisation. Consecutively, this molecular mechanism was proved to enhance formation of filopodia; cell surface projections that allow cell sensing surrounding environment and migrate efficiently. Our results visualize JNK dependent and MARCKSL1 executed induction of filopodia in neurons and fibroblast indicating general mechanism. Subsequently, inactivation of JNK action on MARCKSL1 shifts cellular actin machinery into lamellipodial dynamic arrangement. Tuning of actin cytoskeleton inevitably melds with cell migration. We observed that both active JNK and JNK pseudo-phosphorylated form of MARCKSL1 reduce actin turnover in intact cells leading to overall diminished cell motility. We demonstrate that tumour transformed cells from breast, prostate, lung and muscle-derived cancers upregulate MARCKSL1. We showed on the example of prostate cancer PC-3 cell line that JNK phosphorylation negatively controls MARCKSL1 ability to induce migration, which precedes cancer cell metastasis. The second round of identification of JNK physiological substrates resulted in detection of predominant motor protein kinesin-1 (Kif5). Mass spectrometry detailed analysis showed evident endogenous phosphorylation of kinesin-1 on S176 within motor domain that interacts with MT. In vitro phosphorylation of bacterially expressed kinesin heavy chain by JNK isoforms displayed higher specificity of JNK1 when compared to JNK3. Since, JNK1 is constitutively active in neurons it signified physiological aspect of kinesin-1 regulation. Subsequent biochemical examination revealed that kinesin-1, when not phosphorylated on JNK site, exhibits much higher affinity toward MTs. Expression of the JNK non-phosphorable kinesin-1 mutant in intact cells as well as in vitro single molecule imaging using total internal reflection fluorescence microscopy indicated that the mutant loses normal speed and is not able to move processively into proper cellular compartments. We identify novel kinesin-1 cargo protein STMN2/SCG10, which along with known kinesin-1 cargo BDNF is showing impaired trafficking when JNK activity is inhibited. Our data postulates that constitutive JNK activity in neurons is crucial for unperturbed physiologically relevant transport of kinesin-1 dependant cargo. Additionally, my work helps to validate another novel physiological JNK1 effector STMN2/SCG10 as determinant of axodendritic neurites dynamics in the developing brain through regulation of MT turnover. We show successively that this increased MT dynamics is crucial during developmental radial migration when brain layering occurs. Successively, we are able to show that introduction of JNK phosphorylation mimicking STMN2/SCG10 S62/73D mutant rescues completely JNK1 genetic deletion migration phenotype. We prove that STMN2/SCG10 is predominant JNK effector responsible for MT depolymerising activity and neurite length during brain development. Summarizing, this work describes identification of three novel JNK substrates MARCKSL1, kinesin-1 and STMN2/SCG10 and investigation of their roles in cytoskeleton dynamics and cargo transport. This data is of high importance to understand physiological meaning of JNK activity, which might have an adverse effect during pharmaceutical intervention aiming at blocking pathological JNK action.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Transcriptional and Epigenetic Regulation of Human CD4+ T Helper Lineage Specification

Activated T helper (Th) cells have ability to differentiate into functionally distinct Th1, Th2 and Th17 subsets through a series of overlapping networks that include signaling and transcriptional control and the epigenetic mechanisms to direct immune responses. However, inappropriate execution in the differentiation process and abnormal function of these Th cells can lead to the development of several immune mediated diseases. Therefore, the thesis aimed at identifying genes and gene regulatory mechanisms responsible for Th17 differentiation and to study epigenetic changes associated with early stage of Th1/Th2 cell differentiation. Genome wide transcriptional profiling during early stages of human Th17 cell differentiation demonstrated differential regulation of several novel and currently known genes associated with Th17 differentiation. Selected candidate genes were further validated at protein level and their specificity for Th17 as compared to other T helper subsets was analyzed. Moreover, combination of RNA interference-mediated downregulation of gene expression, genome-wide transcriptome profiling and chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq), combined with computational data integration lead to the identification of direct and indirect target genes of STAT3, which is a pivotal upstream transcription factor for Th17 cell polarization. Results indicated that STAT3 directly regulates the expression of several genes that are known to play a role in activation, differentiation, proliferation, and survival of Th17 cells. These results provide a basis for constructing a network regulating gene expression during early human Th17 differentiation. Th1 and Th2 lineage specific enhancers were identified from genome-wide maps of histone modifications generated from the cells differentiating towards Th1 and Th2 lineages at 72h. Further analysis of lineage-specific enhancers revealed known and novel transcription factors that potentially control lineage-specific gene expression. Finally, we found an overlap of a subset of enhancers with SNPs associated with autoimmune diseases through GWASs suggesting a potential role for enhancer elements in the disease development. In conclusion, the results obtained have extended our knowledge of Th differentiation and provided new mechanistic insights into dysregulation of Th cell differentiation in human immune mediated diseases.

Choosing the target loci : heat shock factors HSF1 and HSF2 as regulators of cell stress and development

Heat shock factors (HSFs) are an evolutionarily well conserved family of transcription factors that coordinate stress-induced gene expression and direct versatile physiological processes in eukaryote organisms. The essentiality of HSFs for cellular homeostasis has been well demonstrated, mainly through HSF1-induced transcription of heat shock protein (HSP) genes. HSFs are important regulators of many fundamental processes such as gametogenesis, metabolic control and aging, and are involved in pathological conditions including cancer progression and neurodegenerative diseases. In each of the HSF-mediated processes, however, the detailed mechanisms of HSF family members and their complete set of target genes have remained unknown. Recently, rapid advances in chromatin studies have enabled genome-wide characterization of protein binding sites in a high resolution and in an unbiased manner. In this PhD thesis, these novel methods that base on chromatin immunoprecipitation (ChIP) are utilized and the genome-wide target loci for HSF1 and HSF2 are identified in cellular stress responses and in developmental processes. The thesis and its original publications characterize the individual and shared target genes of HSF1 and HSF2, describe HSF1 as a potent transactivator, and discover HSF2 as an epigenetic regulator that coordinates gene expression throughout the cell cycle progression. In male gametogenesis, novel physiological functions for HSF1 and HSF2 are revealed and HSFs are demonstrated to control the expression of X- and Y-chromosomal multicopy genes in a silenced chromatin environment. In stressed human cells, HSF1 and HSF2 are shown to coordinate the expression of a wide variety of genes including genes for chaperone machinery, ubiquitin, regulators of cell cycle progression and signaling. These results highlight the importance of cell type and cell cycle phase in transcriptional responses, reveal the myriad of processes that are adjusted in a stressed cell and describe novel mechanisms that maintain transcriptional memory in mitotic cell division.

Novel cancer-related regulatory targets for the signalling sphingolipids sphingosine-1-phosphate and sphingosylphosphorylcholine

The signalling sphingolipid sphingosine-1-phosphate (S1P) is necessary for development of the immune system and vasculature and on a cellular level regulates migration, proliferation and survival. Due to these traits S1P has an important role in cancer biology. It is considered a primarily cancer-promoting factor and the enzyme which produces it, sphingosine kinase (SphK), is often over-expressed in tumours. S1P is naturally present in the blood, lymph, tissue fluids and cell cytoplasm and functions through its cell surface receptors (S1P1-5) and as an intracellular second messenger. Sphingosylphosphorylcholine (SPC) is closely related to S1P and has similar regulatory functions but has not been extensively studied. Both S1P and SPC are able to evoke either stimulatory or inhibitory effects on cancer cells depending on the context. The aim of this thesis work was to study novel regulatory targets of S1P and SPC, which mediate the effects of S1P/SPC signalling on cancer cell behaviour. The investigated targets are the transcription factor hypoxia-inducible factor 1 (HIF-1), the intermediate filament protein vimentin and components of the Hippo signalling pathway. HIF-1 has a central role in cancer biology, as it regulates a multitude of cancer-related genes and is potently activated by intratumoural hypoxia through stabilization of the regulatory subunit HIF-1α. Tumours typically harbour high HIF-1α levels and HIF-1, in turn, facilitates tumour angiogenesis and metastasis and regulates cancer cell metabolism. We found S1P to induce follicular thyroid cancer cell migration in normal oxygen conditions by increasing HIF-1α synthesis and stability and subsequently HIF-1 activity. Vimentin is a central regulator of cell motility and is also commonly over-expressed in cancers. Vimentin filaments form a cytoskeletal network in mesenchymal cells as well as epithelial cancer cells which have gone through epithelial-mesenchymal transition (EMT). Vimentin is heavily involved in cancer cell invasion and gives tumours metastatic potential. We saw both S1P and SPC induce phosphorylation of vimentin monomers and reorganization of the vimentin filament network in breast and anaplastic thyroid cancer cells. We also found vimentin to mediate the anti-migratory effect of S1P/SPC on these cells. The Hippo pathway is a novel signalling cascade which controls cancer-related processes such as cellular proliferation and survival in response to various extracellular signals. The core of the pathway consists of the transcriptional regulators YAP and TAZ, which activate predominantly cancer-promoting genes, and the tumour suppressive kinases Lats1 and Lats2 which inhibit YAP/TAZ. Increased YAP expression and activity has been reported for a wide variety of cancers. We found SPC to regulate Hippo signalling in breast cancer cells in a two-fold manner through effects on phosphorylation status, activity and/or expression of YAP and Lats2. In conclusion, this thesis reveals new details of the signalling function of S1P and SPC and regulation of the central oncogenic factors HIF-1 and vimentin as well as the novel cancer-related pathway Hippo.