Indiana University-Purdue University Indianapolis (IUPUI) / Multiple myeloma (MM) is the most frequent cancer to involve the skeleton, with over 80% of myeloma patients developing lytic bone disease (MMBD). Importantly, MM-associated bone lesions rarely heal even when patients are in complete remission. Bone marrow stromal cells (BMSCs) isolated from MM patients have a distinct genetic profile and an impaired osteoblast (OB) differentiation capacity when compared to BMSCs from healthy donors. Utilizing an in vivo model of MMBD and patient samples, we showed that BMSCs from tumor-bearing bones failed to differentiate into OBs weeks after removal of MM cells. Both Runx2 and Osterix, the master transcription factors for OB differentiation, remained suppressed in these BMSCs. However, the molecular mechanisms for MM-induced long-term OB suppression are poorly understood. We characterized both Runx2 and Osterix promoters in murine pre-osteoblast MC4 cells by chromatin immunoprecipitation (ChIP). The transcriptional start sites (TSSs) of Runx2 and Osterix in untreated MC4 cells were co-occupied by transcriptionally active histone 3 lysine 4 tri-methylation (H3K4me3) and transcriptionally repressive histone 3 lysine 27 tri-methylation (H3K27me3), termed the “bivalent domain”. These bivalent domains became transcriptionally silent with increasing H3K27me3 levels when MC4 cells were co-cultured with MM cells or treated with TNF-α, an inflammatory cytokine increased in MM bone marrow microenvironment. The increasing H3K27me3 levels induced by MM cells or TNF-α were associated with the downregulation of the H3K27 demethylase JMJD3 in MC4 cells and murine BMSCs. Knockdown of JMJD3 in MC4 cells was sufficient to inhibit OB differentiation. Further, ectopic overexpression of JMJD3 in MC4 cells partially rescued the suppression of osteoblast differentiation induced by TNFa. We also found that pre-incubation of MC4 cells with the NF-kB inhibitor quinazoline (QNZ) before TNF-a treatment prevented the downregulation of JMJD3. In agreement with our in vitro findings, BMSCs from MM patients had persistently decreased JMJD3 expression compared to healthy BMSCs. Our findings together demonstrate that decreased JMJD3 expression in BMSCs contributes to the long-term OB suppression in MMBD by remodeling histone landscapes at the Runx2 and Osterix TSSs. Thus, developing strategies to restore JMJD3 expression in BMSCs should increase bone formation and possibly decrease tumor burden in MM.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20078号 / 医博第4171号 / 新制||医||1018(附属図書館) / 33194 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Calcific aortic valve disease (CAVD) is the most common cause of aortic valve failure and replacement in the elderly population, affecting 25% of the population over 65 years of age. Current pharmacological approaches for preventing the onset and progression of calcific aortic valve disease have not shown consistent benefits in clinical studies. Differentiation of valvular interstitial cells (VICs) into osteoblast–like cells is an integral step in the calcification process. Although clinical evidence suggests hypertension as a potential candidate contributing to the development of CAVD, the underlying molecular mechanisms that cause de-differentiation remain unclear. The present study investigates the role of elevated cyclic pressure in modulating osteoblast differentiation pathways in VICs in vitro. We used a combination of systems biology modeling and pathway-based analyses to identify novel genes and molecular mechanisms that are activated in valve tissue during exposure to elevated pressure conditions. Our results show that elevated pressure induces a gene expression pattern in valve tissue that is considerably similar to that seen in CAVD, underlining the key role of hypertension as an initiating factor in the onset of pathogenesis. In addition, our analysis revealed a set of genes that was not previously known to be regulated in valve tissue in a pressure dependent manner. Currently, the molecular mechanisms involved in CAVD and their associations with changes in local mechanical environment are poorly understood, and thus a better understanding of the cell based process mediating CAVD progression will improve our ability to develop potential medical therapies for this disease.

Osteoactivin (OA) is a glycoprotein required for the differentiation of osteoblasts. In osteoblasts, Bone Morphogenetic Protein-2 (BMP-2) activated Smad1 signaling enhances OA expression. However, the transcriptional regulation of OA gene expression by BMP-2 is still unknown. The aim of this study was to characterize BMP-2-induced transcription factors that regulate OA gene expression during osteoblast differentiation. The stimulatory effects of BMP-2 on OA transcription were established by cloning the proximal 0.96kb of rat OA promoter region in a luciferase reporter vector in various osteogenic cell types. Further, by deletion and mutagenesis analyses of the cloned OA promoter, key binding sites for osteogenic transcription factors namely, Runx2, Smad1, Smad4 and homeodomain proteins (Dlx3, Dlx5 and Msx2) were identified and characterized. Utilizing specific siRNAs to knock down Runx2, Smad1, Smad4, Dlx3, Dlx5 or Msx2 proteins in osteoblasts, we found that Runx2, Smad1, Smad4, Dlx3 and Dlx5 proteins up-regulate OA transcription, whereas, Msx2 down-regulated OA gene expression. These specific effects of transcription factors on OA promoter regulation were confirmed by forced expression of transcription factors. Most notably, BMP-2-stimulated cooperative and synergistic interactions between Runx2-Smad1 proteins and Dlx3-Dlx5 proteins that up-regulate OA promoter activity. Electrophoretic mobility shift and supershift assays demonstrated that BMP-2 stimulates interactions between Runx2, Smad1 and Smad4 and homeodomain transcription factors with the OA promoter regions flanking the -585 Runx2 binding site, the -248 Smad binding site and the region between the -852 and the -843 homeodomain binding sites relative to transcription start site. The OA promoter region was occupied by Runx2 and also Dlx3 transcription factors during proliferation stages of osteoblast differentiation. As the osteoblasts progress from proliferation to matrix maturation stages of differentiation, the OA promoter was predominantly occupied by Runx2 and to a lesser extent Dlx5 in response to BMP-2. Finally, during matrix mineralization stages of osteoblast differentiation, BMP-2-induced a robust recruitment of Dlx5, Smad1, Dlx3 and Msx2 proteins with simultaneous dissociation of Runx2 from the OA promoter region. In conclusion, the BMP-2-induced osteogenic transcription factors Runx2, Smad1, Smad4, Dlx3, Dlx5 and Msx2 provide key molecular switches that regulate OA transcription during osteoblast differentiation. / Cell Biology

Osteoarthritis (OA) ist eine chronische Erkrankung der Gelenke des menschlichen Körpers, insbesondere des Kniegelenkes. Sie ist durch entzündliche und degenerative Prozesse gekennzeichnet, die Patienten in ihrer Beweglichkeit stark einschränkt. In der komplexen Pathophysiologie kommt es unter anderem zu zellmorphologischen Veränderungen der knorpelbildenden Zellen, den Chondrozyten, und zu destruktiven Veränderungen der Knorpelmatrix. Bisherige therapeutische Ansätze bestehen in meist in einer rein symptomatischen Therapie durch Schmerzmittel sowie der operativen endoprothetischen Versorgung als Ultima Ratio. Eine kurative Therapie ist bisher nicht möglich. Einen Ansatz für eine kurative Therapie könnte eine Subpopulation der Zellen des Knorpelgewebes bieten. Chondrogene Progenitor Zellen (CPCs) stellen als Vorläuferzellen der Chondrozyten, gesteuert durch das prochondrogene Gen SOX9 und das proosteogene Gen RUNX2, einen möglichen regenerativen Ansatz in der Behandlung dar. Eine Rolle in diesem Prozess könnten die Wachstumsfaktoren TGF- β3 und EGF sowie das Matrixmolekül Biglycan darstellen. In dieser Arbeit konnte gezeigt werden, dass diese Wachstumsfaktoren, deren Rezeptoren und das Matrixmolekül Biglycan im osteoarthrischen Knorpel eine Rolle spielen. Insbesondere konnte in vitro gezeigt werden, dass CPCs unter dem Einfluss dieser Moleküle zu einer vermehrten SOX9 und verminderten RUNX2-Expression angeregt werden. Unter der Hypothese, dass sich CPCs auf diese Art zu Chondrozyten differenzieren lassen und so den Knorpel wiederherstellen, könnten diese Moleküle einen möglichen Baustein einer zukünftigen Therapie der OA darstellen.

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Dentale Implantate sind heutzutage ein fester Bestandteil der modernen Zahnheilkunde. Durch die steigende Anzahl gesetzter Implantate steigt auch die absolute Zahl an Misserfolgen. Der Hauptgrund für den Verlust von Implantaten ist die Periimplantitis. Sie stellt eine Entzündung des Weich- und Hartgewebes um dentale Implantate dar, welche im ultimativen Stadium den Verlust der Integrität des Implantats zur Folge hat. Das Ziel dieser Arbeit soll es sein, einen Überblick über die Genexpression des entzündeten periimplantären Gewebe zu erlangen um so die Periimplantitis auf mikrobiologischer Ebene zu charakterisieren. Um das zu erreichen wurde umgebendes Gewebe von Implantaten mit Periimplantitis und von Implantaten ohne Periimplantitis von menschlichen Patienten gewonnen. Für die Ermittlung der Ergebnisse wurde die Microarray-Technik angewandt (Affymetrix Gene- Chip R Human Gene 1.0 ST Array) und um diese Ergebnisse zu validieren wurde die qPCR-Technik verwendet (real-time-PCR). Die Ergebnisse zeigen, dass klassische Entzündungsmarker wie IL-1, -8 und -6 sind in ihrer Expression stark erhöht. Die erhöhte Expression von CD24 zusammen mit weiteren Cluster of Differentiation und das Expressionsmuster der Interleukine zeigen, dass das unspezifische Immunsystem eine Hauptrolle bei der Entzündung um Implantate spielt. Makrophagen und Monozyten sind sehr wahrscheinlich die am stärksten vertretene Spezies von Abwehrzellen im von Periimplantitis betroffenen Gewebe. Marker für den Abbau von Gewebe, die MMPs-1, -12 und -13 sind in ihrer Expression erhöht, ihre Inhibitoren, die TIMPs sind in ihrer Expression erniedrigt. MMP-12 ist die Makrophagen spezifische Elastase und somit ein weiterer Indikator für die tragende Rolle von Makrophagen bei der Periimplantitis. Kollagen-3 ist das einzige Kollagen, welches den Ergebnissen dieser Arbeit zufolge in seiner Expression erhöht ist. Kollagen-3 findet sich neben Haut- und Muskelzellen in der Wand von Blutgefäßen. Transkriptions- und Wachstumsfaktoren wie RUNX2, SOX2 und FGF18 sind den Ergebnissen dieser Arbeit nach im entzündeten periimplantären Gewebe in ihrer Expression erniedrigt. Fazit: Anhand der Ergebnisse dieser Arbeit lässt sich die Periimplantitis als ein gut durchblutetes, granulationsgewebe-ähnliches-Gewebe mit einer herabgesetzten Kompetenz zur Regeneration charakterisieren. Das unspezifische Immunsystem ist Hauptakteur bei der Periimplantitis und die hauptsächlich anwesenden Immunzellen sind Makrophagen und Monocyten. Die Gewebehomöostase ist in Richtung Gewebeabbau verschoben.

In the United States, metastatic breast cancer kills approximately 40,000 women and 400 men annually, and approximately 200,000 new cases of breast cancer are diagnosed each year. Worldwide, breast cancer is the leading cause of cancer deaths among women. Despite advances in the detection and treatment of metastatic breast cancer, mortality rates from this disease remain high because the fact is that once metastatic, it is virtually incurable. It is widely accepted that a major reason breast cancer continues to exhibit recurrence after remission is that current therapies are insufficient for targeting and eliminating therapy-resistant cancer cells. Emerging research has demonstrated that these therapy-resistant cells possess stem cell-like properties and are therefore commonly referred to as breast cancer stem cells (BCSCs). A major hallmark of BCSCs is the cell surface expression of CD44 and lack of expression of CD24, the so-called CD24-/CD44+ phenotype. Research indicates that this dangerous and rare subpopulation of BCSCs may be responsible for cancer onset, recurrence, and ultimately metastasis that leads to death. Two different model systems were utilized in this research. The first was the MCF7 cell line, a luminal A tumor subtype representative of a mildly invasive breast ductal carcinoma with an ER+/PR+/-/HER2- immunoprofile. The second was the MCF10A breast cancer progression model, which consists of three cell lines: MCF10A, MCF10AT1, and MCF10CA1a. In this system, spontaneously immortalized, non-malignant MCF10A cells were transfected with constitutively active H-Ras to form pre-malignant MCF10AT1 cells, which were then subcutaneously injected into mice and allowed to metastasize in order to form the oncogenic MCF10ACA1a cell line. This thesis presents evidence of a CD24low/-/CD44+ BCSC subpopulation within the MCF10A breast cancer progression model system. Findings indicate that RUNX1 and RUNX2 expression levels are involved in maintaining the BCSC phenotype. Across two different model systems, qRT-PCR analysis revealed that decreased levels of RUNX1 expression and increased levels of RUNX2 expression are essential for the maintenance of the BCSC subpopulation. It was also shown that low expression levels of RUNX1 and high expression levels of RUNX2 are present in CD24low/-/CD44+ BCSCs as compared to CD24+/CD44+ non-BCSCs. Furthermore, shRNA knockdown of RUNX1 was shown to enhance tumorigenicity, while shRNA knockdown of RUNX2 repressed tumorigenicity in BCSCs, as measured by the tumorsphere-formation assay. This research lays the groundwork for future investigations into the roles of RUNX1 and RUNX2 in regulating stemness in breast cancer.

RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.