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The removal of apoptotic cells (AC) can be regarded as an integral component of the program to terminate inflammation. Clearance of AC by professional phagocytes such as macrophages induces an anti-inflammatory phenotype in the latter ones. Anti-inflammatory or M2 polarization is also observed in macrophages infiltrating certain human tumors. These tumor-associated macrophages (TAM) contribute actively to tumor progression by promoting immune evasion, angiogenesis and tumor cell survival. The aim of my Ph.D. thesis was to approach the mechanisms as well as the characteristics of macrophage phenotype alterations induced by AC, and to elucidate a possible connection between tumor cell apoptosis and TAM generation. In the first part of my studies, I investigated the impact of AC on macrophage viability. I could show that macrophage survival against pro-apoptotic agents increased after the interaction with AC. Protection of macrophages against cell death required activation of phosphatidylinositol-3 kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ signaling, and correlated with Bcl-XL and Bcl-2 up-regulation as well as Ser136-Bad phosphorylation. Unexpectedly, neither phagocytosis nor binding of apoptotic debris to the phagocyte was necessary to induce protection. AC released the bioactive lipid sphingosine-1-phosphate (S1P), dependent on sphingosine kinase (SphK) 2, as a survival messenger. These data indicated an active role of AC in preventing cell destruction in their neighborhood. My next aim was to elucidate the mechanism of S1P production by AC. During cell death, SphK 2 was cleaved at its N-terminus by caspase-1. Thereupon, the truncated but enzymatically active fragment of SphK 2 was released from cells. This release was coupled to phosphatidylserine exposure, a hallmark of apoptosis and a crucial signal for the phagocyte/apoptotic cell interaction. Thus, I observed a link between common signaling events during apoptosis and the extracellular production of S1P, which is known to affect immune cell attraction and polarization as well as angiogenesis in cancer. In the next part of my studies, I asked for a correlation between tumor cell apoptosis and TAM polarization. During co-culture of human macrophages with human breast cancer carcinoma cells (MCF-7), the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-α; and interleukin (IL)-12-p70 production, but increased formation of IL-8 and IL-10. Alternative macrophage activation required tumor cell death, because a co-culture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. These phenotype alterations were also achieved with conditioned media from apoptotic tumor cells, which again argued for a soluble factor being involved. Knock-down of SphK2, but not SphK1, to attenuate S1P formation in MCF-7 cells, repressed the otherwise observed alternative macrophage polarization during co-culture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P was characterized by suppression of pro-inflammatory nuclear factor (NF)-κB DNA binding. These findings suggested that tumor cell apoptosis-derived S1P contributes to the macrophage polarization present in human tumors. To validate these in vitro data, I used an in vivo tumor model to clarify the relevance of SphK2 and S1P in tumor development. The growth of, as well as blood vessel infiltration into SphK2 knock-down MCF-7 (MCF-7-siSphK2) xenografts in nude mice was markedly decreased in comparison to control MCF-7 xenografts. In contrast, macrophage infiltration was similar or even more pronounced. These data provided a first hint for an in vivo role of SphK2-derived S1P in macrophage polarization associated with tumor promotion. In summary, these data indicate a new mechanism how AC themselves shape macrophage polarization, which results in the termination of inflammatory responses and macrophage survival. Furthermore, my studies present evidence that human tumors may utilize this mechanism to foster growth via increased angiogenesis.
Aging and age-related diseases are becoming more and more important for our society and our health care system. Alzheimer's disease (AD) is a disorder that destroys some parts of the brain and is characterized by global cognitive decline including a progressive irreversible loss of memory, orientation, and reasoning. “Healthy aging”, therefore, is one of the major aims for modern medicine. Apoptosis, or programmed cell death, plays an important role for example in fetal development, as well as for learning processes. T-lymphocytes usually undergo apoptosis in order to terminate an acute inflammation. The aim of this thesis was to explore the changes in the apoptotic mechanism of peripheral lymphocytes from Alzheimer’s disease (AD) patients in contrast to physiological aging. The experiments were conducted with lymphocytes of healthy volunteers of different ages, AD patients and young and aged mice. Moreover, transgenic mice carrying familiar AD-related mutations were examined. The aging study of peripheral cells of ‘healthy’-aged volunteers revealed an age-related increase of basal apoptosis. In addition, spontaneous apoptosis as well as apoptosis induced by oxidative stress (ROS) or by Fas engagement were enhanced in aging. A closer look at the subcellular basis of the lymphocytes (e.g. B-, NK-, CD4+-, and CD8+-T cells) determined that all lymphocyte subsets were affected by aging. Therefore, it could be concluded that the regulation of apoptosis is generally impaired in lymphocytes of aged persons. The increased susceptibility to oxidative stress supports the ‘Free radical theory of aging’ that claims the radicals to be the cause for the aging-process. In mice an increase of basal, spontaneous and ROS-induced apoptosis was detected in T cells from the spleen, as well. An oral treatment over two weeks with the Ginkgo biloba extract EGb761 showed a clear reduction of ROS-induced apoptosis in the treated group. Interestingly, basal and spontaneous apoptosis, e.g. physiological apoptosis, were not effected by the plant extract. This is an important benefit for therapy since physiological apoptosis has a great relevance in the elimination of cancer-cells for example. In conclusion, the antidementive drug EGb761 reduces specifically ROS-induced apoptosis that a plays an important role in aging as shown in this thesis. Based on the data found in healthy aging, lymphocytes from AD patients were assessed for apoptosis. The cells show enhanced levels of basal, spontaneous, and Fas-induced apoptosis. In subsequent experiments it was demonstrated that mainly the T cells were responsible for the findings. However, the NK-cells provided an important impact as well. In concordance with AD-affected neurons, peripheral lymphocytes of AD patients show clear signs of apoptotic cell death. In addition, basal apoptosis of T cells and the CD4/CD8-ratio showed a correlation with the severity of the dementia. Therefore, it could be speculated that apoptosis is due to activation-induced cell death (AICD) that occurs in acute and chronic activation of adaptive immunity. In AD there is a chronic neuroinflammation in the CNS triggering degeneration of neural tissue. In order to explore this, the experimental model of lymphocyte’s activation was established in healthy aging first. The study included the detection of various events of lymphocyte’s activation on the basis of the T cell subsets (CD4+ and CD8+). The inducibility to mitogenic stimulation clearly decreased in both subsets in aging. In contrast, T lymphocytes from AD patients showed an enhanced activation subsequent to mitogenic stimulation compared with age-matched nondemented persons. Only proliferation of CD8+ T cells was clearly reduced in AD. This data could be clues that an increased generation of memory T cells due to chronic neuroinflammation might be evident in AD. Memory T lymphocytes show increased inducibility upon mitogenic activation. Interestingly, CD8+ memory T cells display decreased prolifertive capacity. Due to activation, cells die by apoptosis later on. It could be concluded that AD patients display an increased amount of memory T cells compared to controls. The data implicate that there could be a cross talk between inflammatory within the brain and inflammatory cells of the periphery. This is an interesting point since the brain used to be assumed as immune-privileged zone. According to the experiment, the information of the diseased brain is transferred to white blood cells. The connection of those two compartments might raise the opportunity to observe and probably to influence easily not-accessible regions like the brain. Transgenic mice carrying mutations in familiar AD-relevant genes (Amyloid-Precursor-Protein, Presenilin-1, respectively) displayed enhanced levels of apoptotic T cells from the spleen, as well. It seems that those mutated proteins influence the regulation of apoptosis. Probably, they are involved in the increased cell death of T- and NK-cells, as well. Animals overexpressing Presenilin-1 showed reduced levels of apoptotic cell death. It was demonstrated with molecuar biology tools that Presenilin-1, processed during apoptosis, has an anti-apoptotic effect.
ADAM15, which belongs to the family of the disintegrin and metalloproteinases, is a multi-domain transmembrane protein. A strongly upregulated expression of ADAM15 is found in inflamed synovial membranes from articular joints affected by osteoarthritis and especially rheumatoid arthritis (RA). During the chronic inflammatory process in RA the synovial membrane gets hyperplastic, resulting eventually in the formation of a pannus tissue, which can invade into the adjacent cartilage and bone thereby destroying their integrity. Previously, the expression of ADAM15 in fibroblasts of the RA synovial membrane was found to confer a significant anti-apoptotic response upon triggering of the Fas receptor, which resulted in the activation of two survival kinases, focal adhesion kinase (FAK) and Src. The Fas receptor, also named CD95, belongs to the death receptor family of the tumor necrosis factor receptors and stimulation of Fas/CD95 by its ligand FasL results in the execution of apoptotic cell death in synovial membranes of RA patients. However, the occurrence of apoptotic cell death in vivo in RA synovial tissues is considerably low despite the presence of FasL at high concentrations in the chronically inflamed joint. Accordingly, a general apoptosis resistance is a characteristic of RA-synovial fibroblasts that contributes considerably to the formation the hyperplastic aggressive pannus tissue. The objective of this study was to investigate the mechanisms underlying the capability of ADAM15 to transform FasL-mediated death- inducing signals into pro-survival activation of Src and FAK in rheumatoid arthritis fibroblasts (RASFs).
In the present study, the down-regulation of ADAM15 by RNA interference resulted in a significant increase of caspase 3/7 activity upon stimulation of the Fas receptor in RASFs. Likewise, chondrocytes expressing a deletion mutant of ADAM15 (ΔC), lacking the cytoplasmic domain, revealed increased caspase activities upon Fas ligation in comparison to cells transfected with full-length ADAM15, clearly demonstrating the importance of the cytoplasmic domain for an increased apoptosis resistance. Furthermore, activation of the Fas receptor triggered the phosphorylation of Src at Y416, which results in the active conformation of Src, as well as the phosphorylation of FAK at Y576/577 and Y861 – the target tyrosines phosphorylated by Src - in full-length ADAM15-transfected chondrocytes. However, cells transfected with ADAM15 mutant (ΔC) or with vector control did not exhibit any activation of Src and FAK upon Fas ligation. This suggested the presence of an as yet unknown protein interaction mediating the Fas triggered activation of the two kinases.
In order to identify this mechanism, the application of signal transduction inhibitors interfering with Calcium signaling either by inhibiting calmodulin with trifluoperazine (TFP) or the Calcium release-activated channel (CRAC/Orai1) with BTP-2 efficiently inhibited the phosphorylation of FAK and Src, revealing a role of calmodulin, the major Ca2+ sensor in cells, in ADAM15-dependent and Fas-elicited activation of the two survival kinases. Also, a direct Ca2+ -dependent binding of calmodulin to ADAM15 could be demonstrated by pull-down assays using calmodulin-conjugated sepharose and by protein binding assays using the recombinant cytoplasmic domain of ADAM15 and calmodulin.
Furthermore, it could be demonstrated in living synovial fibroblasts by double immunofluorescence stainings that triggering the Fas receptor by its ligand FasL or a Fas-activating antibody resulted in the recruitment of calmodulin to ADAM15 as well as to the Fas receptor in patch-like structures at the cell membrane. Simultaneously, Src associated with calmodulin was shown to become engaged in an ADAM15 complex, also containing cytoplasmic-bound FAK, by co-immunoprecipitations.
Additional studies were performed to analyze the efficacy of TFP and BTP-2 on apoptosis induction in synovial fibroblasts from 10 RA patients. Using caspase 3/7 and annexin V stainings for determining apoptosis, it could be shown that both inhibitors did not possess any apoptosis inducing capacity. However, when co-incubated with FasL both compounds synergistically enhanced apoptosis rates in the RASFs. Moreover, an additional silencing of ADAM15 revealed a further significant rise in apoptosis rates upon incubation with FasL/TFP or FasL/BTP-2, providing unequivocal evidence for an involvement of ADAM15 in facilitating apoptosis resistance in RASFs.
Taken together, these results demonstrate that ADAM15 provides a scaffold for the formation of calmodulin-dependent pro-survival signaling complexes upon CRAC/Orai1 coactivation by Fas ligation, which provides a new potential therapeutic target to break the apoptosis resistance in RASFs that critically contributes to joint destruction in RA.
Identification and characterization of TNFalpha responsive genes in human breast cancer cells
(2006)
One of the hallmarks of cancer is the escape of the transformed cells from apoptosis. Therefore, the identification of survival genes, allowing cancer cells to circumvent programmed cell death, could provide new diagnostic markers as well as targets for therapeutic intervention. A well known transcription factor regulating the balance between pro- and anti- apoptotic factors is NF-kappaB, which is strongly induced by tumor necrosis factor alpha (TNFalpha). When cells are stimulated by TNFalpha their response is biphasic with an initial NF-kappaB induction of survival genes which is overridden by the subsequent activation of initiator caspases triggering apoptosis. By combining gene trap mutagenesis with site specific recombination a strategy was developed, which enriches for genes induced by TNFalpha in the human breast cancer cell line MCF-7. The strategy relies on a one way gene expression switch based on Cre/loxP mediated recombination, which uncouples the expression of a marker gene from the trapped cellular promoter thereby enabling the recovery of genes that are only transiently induced by TNFalpha. The marker gene used in these experiments was a dominant negative variant of the TNFalpha-receptor associated protein FADD (dnFADD), which blocks the apoptotic branch of the TNFalpha induced signaling pathway. Initial experiments indicated that MCF-7 cells expressing high levels of dnFADD were insensitive to TNFalpha induced apoptosis and therefore suitable for the installment of a one way gene expression switch susceptible to Cre/loxP mediated recombination. A MCF-7 reporter clone harboring the recombinase dependent gene expression switch was infected with the gene trap retrovirus U3Cre, which inserts the Cre recombinase gene into a large collection of chromosomal sites. Insertion of Cre downstream of an active cellular promoter induces dnFADD expression from the gene expression switch enabling the cells to block TNFalpha triggered apoptosis. From a gene trap integration library containing approximately 2000000 unique proviral integrations, 69 unique TNFalpha inducible gene trap insertion sites were recovered in a two step selection procedure. Sequencing of the genomic regions adjacent to the insertion sites, which were obtained by inverse PCR (gene trap sequence tags, GTSTs), and data base analysis revealed that 42% of the GTSTs belonged to annotated genes, 13% to known cDNAs with open reading frames, 17% to Genscan predicted genes, 9% to ESTs, 9% to repetitive sequences and 10% to unannotated genomic sequence. Overall, 44% of the annotated genes recovered in this screen were directly or indirectly related to cancer, indicating that the gene trap strategy developed here is suitable for the identification of cancer relevant genes. Analysis of the expression patterns of the trapped and annotated genes in wild type cells revealed that 19 out of 24 genes were either up- or down- regulated by a factor of at least 1.45 by TNFalpha. A large fraction of the gene trap insertions were located upstream, in introns or in opposite orientation to annotated transcripts, indicating that the strategy efficiently recovers non-coding RNAs (ncRNAs). While the biological significance of these transcripts still needs to be elucidated, they fall into two main categories. The first category includes gene trap insertions upstream of genes, which could either represent regulatory RNAs interacting with promoter elements or transcripts driven by bidirectional promoters. The second includes inverse orientation gene trap insertions in introns of annotated genes suggesting the presence of natural antisense transcripts (NATs). Interestingly, more than 50% of all antisense integrations are located downstream of transcription start sites predicted by different algorithms supporting the existence of RNAs transcribed from the corresponding genomic regions. Intronic integrations on the coding strand could be derived from cryptic splicing, alternative promoter usage or additional, so far uncharacterized transcripts. Preliminary functional analysis of two genes recovered in this screen encoding the transcription factor ZFP67 and the FLJ14451 protein revealed that FLJ14451 but not ZFP67 inhibited anchorage independent growth in soft agar, suggesting that FLJ14451 might have some tumor suppressor functions. In summary, besides identifying a putative tumor suppressor protein, the present experiments have shown that gene trapping is useful in identifying non-coding transcripts in living cells and may turn out to be the method of choice in characterizing these transcripts whose functions are still largely unknown.
Alzheimer’s Disease (AD) is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterised by the presence of amyloid plaques, intracellular neurofibrillary tangles and pronounced cell death. The aim of this thesis was to investigate pathways involved in the Aß cascade of neurodegeneration. Since novel findings indicate that already this Aß species exerts neurotoxic effects long before hyperphosphorylated tau, neurofibrillary tangles and extracellular Aß plaques appear, the investigations were accomplished with specific regard to the effects of intracellular Aß. The Swedish double mutation in the APP gene results in six- to eightfold increased Aß production of both Aß1-40 and Aß1-42 compared to human wildtype APP cells (APPwt). Data obtained from PC12 cells indicate that it is possible to specifically increase the Aß load without enhancing APP expression levels. On the basis of these findings, it seemed possible to investigate dose-dependent effects of Aß in multiple experimental designs. These assay designs were created in order to mimick different in-vivo situations that are discussed to occur in AD patients: APPsw PC12 cells exhibit low physiological concentrations of Aß within picomolar range in contrast to APPsw HEK cells, expressing Aß levels within the nanomolar range. Of note, the APPsw HEK cells showed a specific and highly significant increase in the intracellular accumulation of insoluble Aß1-42. Moreover, an intracellular accumulation of Aß and APP was found in the mitochondria of the HEK APPsw cells suggesting a direct impact on mitochondrial function on these cells. This effect might finally lead to disturbances in the energy metabolism of the cell or to increased cell death. Furthermore, baseline g- and ß-secretase activity was assessed since these enzymes represent promising therapeutic targets to slow or halt the disease process. As expected, ß-secretase activity was significantly elevated in all APPsw cell lines. This might be due to the proximity of the Swedish double mutation next to the N-terminus of the Aß sequence. Interestingly, g-secretase activity was similarly increased in PC12 APPsw cells. In addition, the toxicity of different Aß species was investigated in SY5Y and PC12 cells with regard to their effect on cellular viability mirrored by mitochondrial activity using MTT assay. Here, it turned out that not monomers, but already dimers are neurotoxic correlates. Fibrillar Aß species showed the highest toxicity. In the next step, SY5Y cells forming endogenous, dimeric APP and Aß were investigated. In accordance with previous findings, these cells showed a decreased MTT reduction potential in comparison to APPwt and control SY5Y cells reflecting a decrease of cellular viability. The impaired energy metabolism of the cells was even more drastically mirrored by reduced baseline ATP levels. In the second part of this thesis, the expression and intracellular distribution of Bcl-2 family proteins and pro-apoptotic mitochondrial factors under baseline conditions and during oxidative stress were analyzed in the APPwt and APPsw bearing cells. The most prominent finding was the reduction of expression levels of the anti-apoptotic factor Bcl-xL in the cytosolic fractions of APPwt and APPsw PC12 cells. This might indicate that a lack of anti-apoptotic factors or their altered intracellular distribution, rather than an increase in caspase-dependent pro-apoptotic factors, could be responsible for the increased vulnerability of APPwt- and APPsw-transfected PC12 cells against oxidative stress. Since total Bcl-xL expression was unaffected in PC12 cells, in contrast to APPwt and APPsw-expressing SY5Y and HEK cells revealing significantly decreased Bcl-xL expression levels. Thus, alterations in Bcl-xL distribution seem to be an early event in the disease process. Increasing Bcl-xL expression might potentially be one promising strategy for AD modification. PC12 and HEK cells bearing APPsw or APPwt were treated with the potent g-secretase inhibitor DAPT. Of note, DAPT did not only efficiently block Aß production, but additionally led to an elevation of the MTT reduction potential, reflecting an increase in cellular viability. As another disease-modifying strategy, several efforts are undertaken to ameliorate AD-relevant symptoms by the treatment with nerve growth factor (NGF). Generally, it is known that substituted pyrimidines have modest growth-promoting effects. Here, KP544, a novel substituted pyrimidine, was characterised. This drug increased MTT reduction potential in terminally differentiated and undifferentiated PC12 cells. Furthermore, treatment with KP544 led to a reduction in Aß1-40 secretion. Thus, one may conclude that the target of KP544, GSK-3ß, represents a connecting link between the two main pathological hallmarks of AD and might thus be a very promising therapeutic target for AD.