Zugl.: Giessen, Universiẗat, Diss., 2008.

Zugl.: Giessen, Universiẗat, Diss., 2008.

Zugl.: Giessen, University, Diss., 2007.

Zugl.: Giessen, University, Diss., 2007.

Anti-Neutrophile Cytoplasmatische Antikörper verursachen nekrotisierende Vaskulitiden kleiner Blutgefäße. Die Serinprotease PR3 ist ein ANCA-Zielantigen, welches von zirkulierenden ANCA auf der Zellmembran erkannt wird. ANCA aktivieren neutrophile Granulozyten, die dann die nekrotisierende Vaskulitis verursachen. Das Membran-PR3 Expressionsmuster ist bimodal wobei mPR3-niedrig- und mPR3-hoch-exprimierende Zellen existieren. Wir testeten die Hypothese, dass ein Membranrezeptor eine hohe mPR3-Expression vermittelt. Wir verwendeten humane neutrophile Granulozyten, neutrophil-differenzierte Stammzellen und transfizierte HEK293 Zellen. Wir identifizierten das Glykoprotein CD177 als einen mPR3-präsentierenden Rezeptor. CD177 zeigte eine spezifische Bindung von reifem PR3-Protein, nicht aber von einem unprozessierten PR3. Wir separierten die mPR3-Zellpopulationen und führten Durchflusszytometrie, Giemsa-Färbung, Western Blot-Experimente und RT-PCR für die PR3 und CD177 mRNA-Expression durch. Wir fanden, dass die mPR3hoch neutrophilen Granulozyten PR3- und CD177-Protein enthielten, während in den mPR3niedrig neutrophilen Granulozyten nur PR3, aber kein CD177 detektierbar war. Die CD177-Regulation vollzog sich auf transkriptioneller Ebene, da die Zellen, die negativ für das CD177-Protein waren auch keine mRNA transkribierten. Um die Grundlage der fehlenden CD177-Transkription zu analysieren, identifizierten wir den Transkriptionsstart von CD177 für eine anschließende Mutations- und SNP-Analyse. Die CD177-Sequenzen der proteinkodierenden Regionen und der Intron-Exon-Übergänge der beiden Zellpopulationen waren identisch. Jedoch fanden wir, dass das CD177-Gen einer monoallelischen Expression unterliegt. Es wurde dabei maternale als auch paternale monoallelische Expression detektiert. In weiterführenden Untersuchungen soll der Regulationsmechanismus der monoallelischen CD177-Expression charakterisiert werden. / Anti-Neutrophil Cytoplasmic Antibodies cause necrotizing small-vessel vasculitis. The serine protease PR3 provides a main ANCA target antigen and is recognized by circulating ANCA on the neutrophil cell surface. ANCA activate neutrophils and activated neutrophils cause vasculitis. The membrane-PR3 expression pattern is bimodal in that low and high mPR3 expressing cells can be distinguished. We tested the hypothesis that a membrane receptor mediates mPR3high expression. We studied human neutrophils, neutrophilic differentiated CD34-positive hematopoietic stem cells and transfected HEK293 cells. We identified the glycoprotein CD177 as an mPR3 presenting receptor. CD177 demonstrated specific binding of mature, but not of unprocessed pro-PR3. We separated the two mPR3 populations and performed cytometry analysis, Giemsa staining, western blot analysis and RT-PCR for PR3 and CD177 expression. We detected PR3 and CD177 protein in mPR3high expressing neutrophils, whereas only PR3, but no CD177 was found in mPR3low expressing cells. Regulation took place on a transcriptional level because cells that were negative for CD177 protein were also negative for mRNA. To further study this finding, we identified the CD177 transcription start for a subsequent mutation and SNP analysis. CD177 sequences of the protein-coding regions and the intron-exon regions did not differ in both populations. However, we found a monoallelic CD177 expression and were able to detect maternal as well as paternal allele expression. Future experiments will elucidate the mechanisms that control monoallelic CD177 gene expression.

Aiming to identify immune molecules with a novel function in cancer pathogenesis, we found the cluster of differentiation 177 (CD177), a known neutrophil antigen, expression to be positively correlated with relapse-free (RFS), metastasis-free (MFS) or overall survival (OS) in several solid cancers including those from breast, prostate, cervix, and lung. To study the role of CD177 in breast cancer, we generated a total body Cd177 knockout mouse. These mice had no profound phenotype at 3 - months of age or younger. The only phenotype found at this age was reduced peripheral neutrophil counts, but no difference in their ability to clear infections. Upon further analysis these mice developed an age dependent hyperproliferative mammary gland phenotype at 10 - months of age that was lost in mice 15 - months and older. Focusing on breast cancer, we found that CD177 is expressed in normal breast epithelial cells and is significantly reduced in invasive cancer. We found that CD177 suppresses breast cancer pathogenesis. To understand the mechanism behind CD177 mediated suppression of breast cancer, we performed mass spectrometry on the purified CD177 complex. Mass spectrometry and co-immunoprecipitation results revealed CD177 interacts with β-Catenin and glycolytic enzymes PFK, aldolase A, GAPDH and enolase-ɑ. Further studies revealed that mechanistically CD177 forms a complex with ECadherin and β-Catenin at adherens junctions. This physical interaction between CD177, E-Cadherin and β-Catenin prevents β-Catenin activation via the canonical WNT. We also found CD177 suppressed WNT/β-Catenin signaling independent of E-Cadherin with an unknown protein. Thus, we identified a novel protein complex involving CD177 and proteins from adherens junctions that can suppress cancer formation via inhibiting the WNT/β-Catenin signaling pathway, a key cellular biological process relevant to the oncogenesis of multiple cancer types and tissue development. The lack of WNT/β- Catenin signaling control explains how mice without CD177 develop hyperproliferation of mammary epithelium in the mouse mammary gland. Interestingly, this phenotype is lost with age, possibly due to a decrease in WNT/β-Catenin signaling resulting from a decrease in progesterone and estrogen. In addition to CD177’s role in the regulation of WNT/β-Catenin signaling we also identified that CD177 plays a role in cancer cell metabolism. Since metabolism plays a significant role in cancer and CD177 interacts with glycolytic enzymes, we sought to determine if CD177 plays a role in metabolism. CD177 appears to interact with glycolytic enzymes, PFK, aldolase A, GAPDH, and ɑ-enolase and ultimately suppresses their mRNA expression. Furthermore, we found novel localization of CD177 at the mitochondrion, thus providing a potential explanation as to how an extracellular membrane bound protein such as CD177 interacts with glycolytic enzymes. Metabolic analysis of CD177 expression on cancer cells revealed that CD177 leads to a decrease in glucose uptake and a slight decrease in basal glycolysis, but an increase in lactate concentration. Further metabolic profiling also revealed that CD177 expression results in a significant decrease in glycolytic capacity (ECAR). Expression of CD177 also resulted in a significant decrease in basal respiration, ATP production, maximal respiration, and spare capacity (OCR) as well as an increase in reactive oxygen species. These data reveal that CD177 plays a novel role in cancer cell metabolism.