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Perlecan is the major heparan sulfate proteoglycan (HSPG) in growth plate cartilage and is critical for growth plate chondrocyte proliferation and proper skeletal development. Its core protein and attached chondroitin sulfate (CS) and heparan sulfate (HS) chains mediate interactions with many diverse proteins. Fibroblast growth factor (FGF)-2 and FGF-18 are other regulators of chondrocyte proliferation in the growth plate. Additionally, FGF-18 controls the hypertrophy and cartilage vascularization necessary for endochondral ossification. The research presented in this dissertation aimed to identify known and novel perlecan-binding proteins that are endogenous to the growth plate and to characterize their interactions with perlecan. FGF-2 (known to bind HSPGs) bound to perlecan in both a cationic filtration (CAF) assay and an immunoprecipitation (IP) assay primarily via the HS chains on perlecan. When digested with chondroitinase ABC to remove its CS chains, perlecan augmented binding of FGF-2 to the FGFR-1 and FGFR-3 receptors and increased FGF-2 -stimulated proliferation in BaF3 cells expressing these FGF receptors. Thus, growth plate perlecan binds to FGF-2 by its HS chains but can only deliver FGF-2 to FGF receptors when its CS chains are removed. FGF-18 (known to bind to heparin and to heparan sulfate from some sources) bound to growth plate perlecan. This binding was unchanged by chondroitinase or heparitinase digestion of perlecan, indicating that perlecan GAGs are not involved in FGF-18 binding. FGF-18 bound equally to recombinant domains I-III of perlecan (Alt1) and to full-length perlecan purified from the growth plate. Additionally, FGF-18 bound equally to recombinant domain III of perlecan, to Alt1 and to Alt2 (a domain I-III variant with no heparan sulfate). Therefore, binding sites for FGF-18 are present in domain III of perlecan. Affinity chromatography isolated histone H3 as a perlecan-binding protein from the chondrocyte matrix. CAF assays confirmed the interaction as specific, dependent primarily on HS chains of perlecan, although CS chains and the perlecan core were also involved. Immunohistochemistry detected perlecan and histone H3 colocalized in growth plate cartilage. These results can help us better understand the growth factor-independent control that perlecan exerts on endochondral ossification and, therefore, long bone growth.

Klotho (KL), an anti-aging protein, attracted interest in the aging field because of the dramatic phenotype of KL deficient mice and its connection to signaling pathways implicated in aging. The KLVS variant consists of the F352V (KLV) and C370S (KLS) substitutions. It was detected in genome wide association studies (GWAS) that linked it to alterations in longevity and disease risk. The molecular mechanism(s) underlying these associations are unknown. To understand how KL increases the risk of age-related diseases, the studies in this dissertation investigated whether expression of the KLVS variant, when compared to wildtype (KLWT), displays differences in processing, protein-protein interactions and enzymatic activity. Differences in processing were evaluated by studying changes in shedding, half-life and plasma membrane localization of KL variants. The decrease in KLV shedding, as measured by the intracellular: extracellular ratio, were explained by a decreased half-life. This decreased half-life is potentially due to decreased KLV plasma membrane localization, which is attenuated by co-expression of dominant negative dynamin, suggesting a role of endocytosis in these differences. To assess whether there are changes in KLVS protein-protein interactions, differences in dimerization were measured by Blue Native gel electrophoresis and cross-linking. KLV dimerization was increased while KLS and KLVS variants decreased dimerization. Co-immunoprecipitation of tagged KL assessed whether these changes were due to alterations in homodimerization. The presence of KLVS in dimers decreased the levels of immunoprecipitated KL suggesting KLVS decreases homodimerization. Changes in heterodimerization of KLVS with fibroblast growth factor receptor (FGFR) 1c were also investigated through co-immunoprecipitation. KLVS increased heterodimerization with FGFR1c. Addition of FGF23, for which KL is a co-receptor, showed that KLVS increases FGF signaling downstream of FGFR1c. To determine differences in enzymatic activity of KLVS, 4-metylumbelliferyl-beta-D-glucuronide was used to measure alterations in glucuronidase activity. Results showed that KLVS had decreased enzymatic activity compared to KLWT. These findings are the first to show that KLVS leads to differences in function as demonstrated by decreased homodimerization and enzymatic activity and increased heterodimerization with FGFR1c. Given the association of KLVS with disease and longevity, these results suggest that these functions are integral in KL's anti-aging role in humans.

Comprehensive genomic profiling has an emerging role in cancer therapeutics. As treatment options remain needed for advanced cancers, patients are relying increasingly more on tumor genomic alterations as possible targets for cancer treatment. Frequent tumor fibroblast growth factor receptor (FGFR) alterations are seen in many cancers, and include genetic amplifications, mutations, rearrangements and fusions. FGFR inhibitors target these receptor alterations and show promise as a drug class. Currently 2 medications are currently FDA approved: erdafitinib and pemigatinib. Through the FDA accelerated approval process, erdafitinib is indicated to treat metastatic urothelial carcinoma with FGFR2 and FGFR3 alterations, whereas pemigatinib is indicated to treat unresectable cholangiocarcinoma with FGFR2 alterations. Despite growing knowledge about such advanced cancers, treatment is usually palliative. With multiple FGFR inhibitors in the pipeline, further FDA approvals are possible, and it is likely their role in therapy will extend to other cancer types. This review outlines erdafitinib, pemigatinib, their role in cancer, as well as outlining the possible future use of other FGFR inhibitors in urothelial carcinoma, cholangiocarcinoma, and other malignancies.

Comprehensive genomic profiling has an emerging role in cancer therapeutics. As treatment options remain needed for advanced cancers, patients are relying increasingly more on tumor genomic alterations as possible targets for cancer treatment. Frequent tumor fibroblast growth factor receptor (FGFR) alterations are seen in many cancers, and include genetic amplifications, mutations, rearrangements and fusions. FGFR inhibitors target these receptor alterations and show promise as a drug class. Currently 2 medications are currently FDA approved: erdafitinib and pemigatinib. Through the FDA accelerated approval process, erdafitinib is indicated to treat metastatic urothelial carcinoma with FGFR2 and FGFR3 alterations, whereas pemigatinib is indicated to treat unresectable cholangiocarcinoma with FGFR2 alterations. Despite growing knowledge about such advanced cancers, treatment is usually palliative. With multiple FGFR inhibitors in the pipeline, further FDA approvals are possible, and it is likely their role in therapy will extend to other cancer types. This review outlines erdafitinib, pemigatinib, their role in cancer, as well as outlining the possible future use of other FGFR inhibitors in urothelial carcinoma, cholangiocarcinoma, and other malignancies.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25490号 / 医博第5090号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 江藤 浩之, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Caenorhabditis elegans is a powerful tool to study cellular migration and morphogenesis during organ development. During pharynx development, the dorsal gland cell, g1p, is born in the anterior aspect of the pharyngeal primordium and undergoes a form of morphogenesis called retrograde extension. egl-15, the single Fibroblast Growth Factor Receptor (FGFR) in C. elegans and ina-1, one of two α-integrin receptors, are both required for the proper extension or migration of g1p cell. Mutations in either egl-15 or ina-1 show similar gland cell over-migration defects where the gland cell body migrates past the terminal bulb and is located in proximity of the intestine. The kinase domain of EGL-15 was found to be required for migration and transgenic rescue strategies were used to determine the tissue of EGL-15 function. RNA interference was used to determine if egl-15 and ina-1 are functioning in the same pathway to regulate gland cell migration. / February 2015

Studies show that fibroblast growth factors (FGFs) control variety of cellular activities such as mitosis, cell differentiation, survival and angiogenesis. The FGF family consists of 23 different heparin-binding proteins. One of the most intensively studied members is human FGF-1 (hFGF-1) because of its critical role in the formation of blood vessels and cell proliferation in some types of cancer. The biological activities of FGFs are primarily mediated via interactions with fibroblast growth factor receptors (FGFR) and are a potent target in cancer. In this study, we report an efficient affinity column purification of hFGF-1 and the D2 domain of FGFR-2 from bacterial expression followed by SDSPAGE analysis. Steady state fluorescence results showed that both proteins were in their native conformation. The 1 H-15N HSQC NMR analysis of hFGF-1 was further performed. The data confirmed the purity and well-conserved native state of the protein. The findings of this study can be used in designing hFGF-1 antagonists with competitive inhibition characteristics. These antagonists could result in possible inhibition of hFGF-1 cell proliferation and angiogenesis associated in tumorigenesis.

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