Intervertebral discs (IVD) degenerate earlier than many other musculoskeletal tissues and will continue to degenerate with aging. IVD degeneration affects up to 80 percent of the adult population and is a major contributing factor to low back pain. Anti-sclerostin antibody is an FDA-approved treatment for osteoporosis in postmenopausal women at high-risk for fracture and, as a systemic stimulant of the Wnt/LRP5/b-Catenin signaling pathway, may impact the IVD. Stabilization of b-Catenin in the IVD increases Wnt signaling and is anabolic to the extracellular matrix (ECM), while deletion of b-catenin or LRP5 decreases Wnt signaling and is catabolic to the ECM. Here, we hypothesized that a reduction of Sost would stimulate ECM anabolism. Lumbar and caudal (tail) IVD and vertebrae of Sost KO and WT (wildtype) mice (n=8 each) were harvested at 16 weeks of age and tested by MRI, histology, immunohistochemistry, Western Blot, qPCR, and microCT. Compared to WT, Sost KO reduced sclerostin protein and Sost gene expression. Next, Sost KO increased the hydration of the IVD and the proteoglycan stain in the nucleus pulposus and decreased the expression of genes associated with IVD degeneration, e.g., heat shock proteins. However, deletion of Sost was compensated by less unphosphorylated (active) b-Catenin protein in the cell nucleus, upregulation of Wnt signaling inhibitors Dkk1 and sFRP4, and catabolic ECM gene expression. Consequently, notochordal and early chondrocyte-like cells (CLCs) were replaced by mature CLCs. Overall, Sost deletion increased hydration and proteoglycan protein content, but activated a compensatory suppression of Wnt signaling that may trigger chondrogenesis and may potentially be iatrogenic to the IVD in the long-term.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21224号 / 生博第393号 / 新制||生||52(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 米原 伸, 教授 上村 匡 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Granulocytes represent the first line of defense against bacteria and fungi. Daily production of granulocytes is sustained by steady state granulopoiesis but under stress (e.g., bacterial infection) this program switches to emergency granulopoiesis (EG) which ensures the production of granulocytes at enhanced and accelerated rates. Very little is known about the regulation of EG. In this thesis, we showed that disruption of the β-catenin-TCF/LEF mediated transcription impairs EG in vivo. Further, we demonstrated that lipopolysaccharide (LPS) administration in mice induces accumulation of active β-catenin in hematopoietic stem and progenitor cells (HSPCs) as early as 4 hours (H) after stimulation, with highest increase at 24H. This effect was at least partially mediated in a niche independent manner, since LPS stimulation in vitro induced β-catenin accumulation in c-Kit+ cells after 2H, with a peak activation at 4H. Using single cell RNA sequencing, we determined the cell cluster dynamics of HSPCs following 4H LPS stimulation. Interestingly, we identified a possible upstream activator of β- catenin in one of the clusters - Wnt10b. Indeed, Wnt10b showed a similar expression pattern as EG master regulator Cebpb and β-catenin activation, following in vitro treatment with LPS. Altogether, our data point...

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Primary cilia are nonmotile, hair-shaped organelles that extend from the basal body in the centrosome. The present study is the first investigation of this organelle in the oligodendrocyte lineage in vivo. I used immunohistochemical approaches in normal and cilia-deficient mutant mice to study cilia in relation to oligodendrogenesis and myelination. Primary cilia immunoreactive for Arl13b and ACIII were commonly present in NG2+ oligodendrocyte progenitor cells (OPCs), in which cilia-associated pathways control proliferation, differentiation, and migration. The loss of primary cilia is generally associated with enhanced Wnt/β-catenin signaling, and Wnt/β-catenin signaling has been shown to promote myelin gene expression. I examined whether the lack of cilia in the oligodendrocyte lineage is associated with elevated Wnt/β-catenin activity. I found that absence of a primary cilium was associated with with higher levels of TCF3, and with β-galactosidase in Axin2-lacZ Wnt reporter mice. This evidence supports the proposal that cilia loss in oligodendrocytes leads to enhanced Wnt/β-catenin activity, which promotes myelination. Cilia are dependent on the centrosome, which assembles microtubules for the cilium, the cytoskeleton, and the mitotic spindle. Centrosomes are the organizing center for microtubule assembly in OPCs, but this function is decentralized in oligodendrocytes. I found that the intensity of centrosomal pericentrin was reduced in oligodendrocytes relative to OPCs, and γ-tubulin was evident in centrosomes of OPCs but not in mature oligodendrocytes. These decreases in centrosomal proteins might contribute to functional differences between OPCs and oligodendrocytes. The importance of cilia in the oligodendrocyte lineage was examined in Tg737orpk mice, which have a hypomorphic IFT88 mutation resulting in decreased cilia numbers and lengths. These mice showed marked, differential decreases in numbers of oligodendrocytes and myelin, yet little or no change in OPC populations. It appears that sufficient cells were available for maturation, but lineage progression was stalled. There were no evident effects of the mutation on Wnt/β-catenin. Factors that might contribute to the abnormalities in the oligodendrocyte lineage of Tg737orpk mice include decreased cilia-dependent Shh mitogenic signaling and dysregulation in cilia-associated pathways such as Notch and Wnt/β-catenin.

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Cirka 300 000 individer drabbas årligen i världen av oral cancer och mer än nittio procent av alla orala cancerformer utgörs av skivepitelcancer. Den femåriga prognosen är generellt 50 % och den 5-åriga relativa överlevnaden har under en tioårsperiod förblivit densamma. Detta motiverar utvecklingen av bättre prognostiska markörer och diagnostiska metoder för att tidigt identifiera de patienter som har risk att utveckla oral skivepitelcancer för att förbättra prognosen och minska lidandet genom tidig insatt behandling. β-catenin är en adhesionsmolekyl som är viktig för bibehållandet av cellulär integration och avvikelser i celladhesionsmolekyler tros spela en central roll när tumörceller invaderar närliggande vävnad det vill säga metastaserar till andra organ.Syftet med studien är att med hjälp av immunohistokemi undersöka om β-catenin kan fungera som en prognostisk markör för utvecklingen av oral skivepitelcancer. Detta görs genom att jämföra förekomsten av β-catenin med hjälp av monoklonala antikroppar i normalt skivepitel, dysplasi och cancer från 18 patienter som har diagnostiserats med oral skivepitelcancer. Infärgningen av Beta catenin jämfördes i normalt oralt skivepitel med cancer och dysplasi för alla biopsier för att undersöka om det förekommer någon skillnad av infärgningen. Förutom detta skedde även en jämförelse av normalt skivepitel med dysplasi och cancer inom varje enskild biopsi.Resultaten visade att det finns en skillnad i uttrycket av β-catenin i normalt skivepitel jämfört med dysplasi och cancer i denna patientgrupp. I denna studie visade mer än 70 % av biopsierna en stark eller måttlig och stark infärgning av β-catenin i normalt skivepitel, mer än 60 % av biopsierna visade en måttlig eller måttlig och svag infärgning av dysplasi och 58,8 % av alla biopsier visade svag infärgning eller ingen och svag infärgning av skivepitelcancer. Då studien visar att mängden av β-catenin är starkast i normalt oralt skivepitel, måttligt i dysplasi och svagast i cancer tyder detta på att β-catenin skulle kunna vara en viktig faktor i utvecklingen av skivepitelcancer i munhålan vilket stämmer väl överens med resultat från andra studier. / Approximately 300,000 individuals are affected every year in the world of oral cancer and more than ninety percent of all oral cancers consists of squamous cell carcinoma. The five-year prognosis is generally 50 % and the 5-year relative survival has over ten years remained the same. This motivates the development of better prognostic markers and diagnostic methods for the early identification of patients at risk of developing oral squamous cell carcinoma to improve prognosis and reduce the suffering of these patients with early treatment.β-catenin is an adhesion molecule that is important for the maintenance of cellular integration and abnormalities of cell adhesion molecules is thought to play a central role in tumorigenesis. The abnormalites is though to enhance tumour cells to break loose from neighbouring cells and invade nearby tissues and organs, however the exact mechanisms are unknown. The purpose of the study is that using immunohistochemistry to examine whether β-catenin may serve as a prognostic marker for the development of oral squamous cell carcinoma. This is done by examining the presence of β-catenin with monoclonal antibodies in 18 biopsies with normal squamous epithelia, dysplasia and cancer from 18 patients diagnosed with oral squamous cell carcinoma from the department of Oral Pathology at Malmö Högskola, Malmö. The staining of beta catenin was compared in normal oral squamous cancer and dysplasia for all biopsies to see whether there is any difference of dyeing. Besides this, there was also a comparison of normal squamous epithelium with dysplasia and cancer in each biopsy.The results showed that there is a difference in the expression of β-catenin in normal squamous epithelium, dysplasia and cancer in this population. In this study, more than 70 % of the biopsies expressed a strong or moderate and strong staining of β -catenin in normal oral squamous epithelium, more than 60 % of the biopsies showed a moderate or moderate and weak staining of dysplasia and 58.8 % of all biopsies showed weak or no staining and weak staining of squamous cell carcinoma.As the study shows that the amount of β -catenin is strongest in normal oral squamous epithelium, moderate in dysplasia and weakest in cancer, this suggests that β -catenin could be an important factor in the development of squamous cell carcinoma of the oral cavity which is in line with results from other studies.

Patients on peritoneal dialysis (PD) are reliant on the peritoneum to provide a semi-permeable barrier to allow for dialysis (solute clearance), salt and water removal (ultrafiltration). PD patients are at risk of developing peritoneal fibrosis and angiogenesis which can lead to a decline in peritoneal membrane function. Specifically, PD patients develop increased solute transport and decreased osmotic conductance leading to ultrafiltration failure. Peritoneal angiogenesis is the leading factor that results in augmented peritoneal membrane solute transport which is associated with worse outcomes – increased risk of mortality and PD technique failure. Transforming growth factor beta (TGFB) is one of the primary cytokines involved in inducing epithelial to mesenchymal transition (EMT) and fibrosis. We hypothesize that PD leads to injury of the epithelial lining of the peritoneum – the mesothelial cells. These cells undergo a transition process and transitioned mesothelium are a source for angiogenic and fibrogenic growth factors. Matrix Metalloproteinase (MMP) 9 is an angiogeneic factor and has been observed to correlate with increased expression of vascular endothelial growth factor (VEGF). MMP9 has the ability to cleave and activate membrane bound factors such as E-cadherin and b-catenin respectively. There is substantial evidence that the canonical WNT/b-catenin pathway is active during fibrosis, and angiogenesis in different biological contexts. Thus, we investigated the role of MMP9 and WNT signaling in peritoneal angiogenesis. Limited evidence exists describing the role of noncanonical WNT signaling but some reports suggest that non-canonical WNT signaling inhibits WNT/b-catenin signaling. Non-canonical WNT5A has differential effects based on receptor context and has been shown to block WNT/b-catenin signaling in the presence of Receptor Tyrosine Kinase Like Orphan Receptor 2 (Ror2). The overall hypothesis of this PhD thesis is that MMP9 and WNT signaling play a key role in inducing peritoneal angiogenesis and are associated with changes in peritoneal membrane function. We expect WNT5A and Ror2 to protect against peritoneal membrane injury. From the overnight effluent of stable PD patients, we cultured mesothelial cells and assayed these for expression of MMP and WNT related genes. MMP9 and WNT1 gene expression were observed to be strongly correlated with peritoneal membrane solute transport in patients on PD. WNT2 mRNA was also positively correlated with peritoneal solute transport. We overexpressed MMP9 in the mouse peritoneum to demonstrate its role in angiogenesis and confirmed these findings using MMP9 -/- mice. In addition to this, we have shown a novel mechanism by which MMP9 induces angiogenesis by E-cadherin cleavage and b-catenin mediated signaling. The observed cross-talk between MMP9 and b-catenin prompted investigation of the activation of canonical WNT/b-catenin signaling in development of peritoneal membrane injury. In an experimental model of TGFB induced pertioneal injury, we confirmed the activation of WNT/b-catenin signaling. In addition to this we, we blocked the WNT pathway and observed that WNT/b-catenin signaling is required to induce peritoneal angiogenesis. WNT5A mRNA was downregulated during TGFB induced injury suggesting a more protective role. Furthermore, several studies have demonstrated its ability to antagonize the WNT/b-catenin signaling pathway. We demonstrated that WNT5A protected against angiogenesis by blocking the canonical WNT pathway. WNT5A is thought to antagonize the WNT/b-catenin signaling pathway by signaling through receptor Ror2. In cell culture, we overexpressed TGFB and blocked Ror2. This resulted in elevated levels of VEGF and fibronectin suggesting that Ror2 is involved in mediating protection. Therefore, Ror2 possesses the ability to regulate VEGF and may be a potential candidate by which WNT5A mediates its protective effects. In conclusion, our findings identified MMP9 and WNT1 as potential biomarkers of increased peritoneal solute transport in patients that are on PD. We have also found a novel mechanism by which MMP9 interacts with b-catenin to induce peritoneal angiogenesis and have provided a first look at WNT/b-catenin signaling in peritoneal angiogenesis. Lastly, we have shown WNT5A to protect against peritoneal angiogenesis. Taken together, our findings are not only significant to the realm of PD research but hold wide applicability to research in the biomedical sciences. / Thesis / Doctor of Philosophy (PhD)