Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

Lysophosphatidic acid (LPA) is the simplest of the glycerol lipids and regulates a number of cellular processes such as morphological changes, migration, proliferation, and inhibition of apoptosis. LPA exerts these effects through activation of the G-protein coupled receptors (GPCRs) LPA1-6 and the intracellular fatty acid receptor peroxisome proliferator-activated receptor-gamma (PPARγ). The overall goal of this thesis was to determine the mechanisms by which LPA enhances cell survival by inhibiting apoptosis. The project was divided into three studies: 1) to determine the mechanism of LPA-mediated inhibition of p53 in A549 lung carcinoma cells, 2) to investigate the regulation of growth plate chondrocytes by LPA, and 3) to determine the mechanisms of LPA-mediated effects in the growth plate. In the first study, evidence is provided that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells. The LPA effect depends upon increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. The result of LPA-mediated inhibition of p53 in A549 cells is enhanced resistance to chemotherapeutic-induced apoptosis. In the second study, the role of LPA in resting zone chondrocytes (RC cells) was investigated. RC cells are regulated by 24,25-dihydroxyvitamin D3 [24,25(OH)[subscript2]D [subscript 3]] via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)[subscript 2]D[subscript 3] stimulates rat costochondral RC cells to release LPA. Additionally, we demonstrated that RC cells respond to LPA with increased proliferation, maturation, and inhibition of apoptosis. In the final study, the mechanism of LPA and 24R,25(OH)[subscript 2]D[subscript 3]-mediated inhibition of chondrocyte apoptosis was further investigated. Our data show that 24R,25(OH)[subscript 2]D[subscript 3] inhibits apoptosis through Ca⁺⁺, PLD, and PLC signaling and through LPA/Gαi/PI[subscript 3]K/mdm2-mediated degradation of p53, resulting in decreased caspase-3 activity. Collectively, our data establish LPA, vitamin D, and p53 as an anti-apoptotic signaling axis.

Growth plate is a highly organized cartilaginous tissue found at the end of long bones and is responsible for longitudinal growth of the bones. Growth plate fracture leads to retarded growth and unequal limb length, which might have a lifelong effect on a person’s physical stature. This research is a tissue engineering approach for the treatment of growth plate injury. Insulin-like growth factor I (IGF-I), which can stimulate cartilage formation, was encapsulated within PLGA microspheres that were then used to form porous scaffolds. The release profile of the IGF-I from the PLGA scaffold showed a biphasic release pattern. In vitro studies were done by seeding rat bone marrow cells (BMCs) on the top of IGF-I encapsulated PLGA scaffolds, and the results showed an increase in cell multiplication and glycosaminoglycan content. The final in vivo studies were conducted by creating growth plate injury and implanting scaffolds in the tibiae of the New-Zealand white rabbits. Histological analysis of tissue sections showed regeneration of cartilage, albeit with disorganized structure, at the site of implantation of IGF-I encapsulated scaffolds. This work will be a significant step towards tissue engineering of growth plate cartilage.

Magnetresonanztomographische Studie zur altersabhängigen Abbildung der Wachstumsknorpel des distalen Radius des Pferdes unter besonderer Berücksichtigung des Epiphysenfugenknorpels. Es wurden magnetresonanztomographische Untersuchungen von 28 Gliedmaßenabschnitten des distalen Radius im Alter von zwei Tagen bis 17 Jahren durchgeführt. Die Studie wurde an einem 1,5 Tesla Magnetom “Symphony“ (Siemens) in vier unterschiedlichen Sequenzen (T1-gewichtet T1w, T2-gewichtet T2w, Protonendichte PD, T2 Double Echo in Steady State T2-dess) und zwei Schnittebenen (dorsal, sagittal) durchgeführt. Die Darstellung der knorpeligen Wachstumsregionen des distalen Radius mit besonderem Hinblick auf seine Epiphysenfuge wurde deskriptiv erfasst und altersbedingte Unterschiede definiert. Die durchschnittliche Dicke des sich darstellenden Epiphysenfugenknorpels wurde in zwei Sequenzen (T1w und T2-dess) vermessen und in Bezug zu dem ansteigenden Alter des Probenmaterials gesetzt. Die Proben konnte man fünf Gruppen zuordnen. In Gruppe 1 konnten sowohl der Wachstumsknorpel der distalen Ossifikationszentren als auch die knorpeligen Anteile der Epi- und Apophysenfugen dargestellt werden. In der Gruppen 2 ließen sich die Apo- und Epiphysenfugen darstellen, in Gruppe 3 nur die Epiphysenfugen. Gruppe 4 beschrieb partiell geschlossene Epiphysenfugen und in Gruppe 5 stellten sich nur 88 noch Fugennarben dar. Eine alterskorrelierende Abnahme der mittleren Knorpeldicke der Epiphyse konnte mittels Vermessungen der Knorpelschichten nachgewiesen werden. Der hyaline Knorpel war mit den gewählten Sequenzen sehr gut beurteilbar. Der Wachstumsknorpel der Epi- und Apophysen stellte sich in den T1w und PD mit hell-intermediärer und in den T2w mit intermediärer Signalintensität dar. Die knorpeligen Anteile der Epi- und Apophysenfuge wurden in T1w intermediär bis hellintermediär, in T2w hell-intermediär und in der PD hell-intermediär bis hyperintens dargestellt. Angrenzende Strukturen wie die subchondrale Knochenplatte und die Mineralisationszone der Epiphysenfuge konnten in dunkel-intermediären bis hypointensen Signalen abgebildet werden. Die Magnetresonanztomographie (MRT) hat sich als geeignetes Verfahren erwiesen, die knorpeligen Strukturen der Wachstumsregion des distalen Radius des Pferdes bildlich wiederzugeben. Altersabhängige strukturelle Unterschiede konnten im MRT dargestellt werden. Damit leistet die vorgestellte Studie einen wichtigen Beitrag über die anatomischen Verhältnisse und deren physiologische Darstellung.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23834号 / 薬科博第149号 / 新制||薬科||16(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 金子 周司, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 168 pages. Includes vita. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references. Also available online.

Thesis (M.S.)--University of Missouri--Columbia, 2003. / "December 2003." Typescript. Vita. Includes bibliographical references (leaves 84-96). Also issued on the Internet.