The isoprenoid biosynthetic pathway is targeted in the treatment of several diseases, including hypercholesteremia and bone related disorders. Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are isoprenoid biosynthetic pathway intermediates that are utilized during post-translational modification of proteins termed farnesylation and geranylgeranylation, respectively, together known as prenylation. The Ras and Rho GTPase family members are examples of proteins that are prenylated. Prenylation is essential for proper membrane localization and function of these small GTPases. Activating mutations or over-expression of these proteins promote oncogenic events, such as increased proliferation and migration. Studies have demonstrated that farnesyl transferase inhibitors and geranylgeranyl transferase inhibitors possess anti-cancer effects in humans and animal models of cancer, respectively. An alternative way to impair protein prenylation is through the depletion of FPP and GGPP. Statins and nitrogenous bisphosphonates (NBPs) deplete FPP and GGPP leading to impaired protein prenylation by inhibiting HMG-CoA Reductase (HMGCR) and FPP synthase (FDPS), respectively. These drugs have been shown to induce apoptosis, inhibit cancer cell migration, and induce cell cycle arrest. The anti-cancer effects of statins and NBPs can be prevented by GGPP addition, suggesting that GGPP depletion may be the mechanism by which these agents interfere with cancer cell survival. We and our collaborators have developed bisphosphonate inhibitors of GGPP synthase (GGDPS), an enzyme that produces GGPP from the substrates FPP and isopentenyl pyrophosphate. The goal of this research was to identify novel GGDPS inhibitors and to assess the effects of specific inhibition of GGDPS on cancer cell survival and function. Two aromatic bisphosphonates were identified as potent inhibitors of GGDPS in enzyme and cellular assays. Apoptosis hallmarks such as PARP cleavage and DNA fragmentation demonstrated that GGDPS inhibition induces apoptosis in K562 chronic myeloid leukemia cells through GGPP depletion and FPP accumulation. Isobologram analysis and enhanced impairment of protein geranylgeranylation showed that GGDPS inhibition is synergistic with the inhibition of HMGCR. Migration assays, transwell assay and large scale digital cell analysis system microscopy, demonstrated that GGDPS inhibition interferes with MDA-MB-231 breast cancer cell migration. Increased LC3-II expression showed that FDPS and GGDPS inhibition induces autophagy in PC3 prostate and MDA-MB-231 breast cancer cells. Inhibition of autophagy enhances the toxic effects of GGDPS inhibition as measured by MTT assay. Propidium iodine staining of DNA and immunostaining of cell cycle proteins such as p27 did not show significant effects of GGDPS inhibition on cell cycle progression. Importantly, exogenous addition of GGPP prevented most of the effects observed with GGDPS inhibition, suggesting specific inhibition of GGDPS by our bisphosphonate inhibitors. The data obtained herein suggest that GGDPS can be targeted to interfere with the progression of cancer cells.

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. By inhibiting HMG-CoA reductase (HMGCR) statins deplete the cellular isoprenoid biosynthetic pathway products farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Current thought in the field is that statins stimulate bone formation through the depletion of GGPP, since exogenous GGPP prevents the effects of statins on osteoblasts in vitro. We hypothesized that direct inhibition of GGPP synthase (GGPPS) would similarly stimulate osteoblast differentiation. Digeranyl bisphosphonate (DGBP), a specific inhibitor of GGPPS, decreased GGPP levels in MC3T3-E1 pre-osteoblasts and calvarial osteoblasts leading to impaired protein geranylgeranylation. In contrast to our hypothesis, DGBP inhibited the matrix mineralization of MC3T3-E1 cells and the expression of osteoblast differentiation markers in calvarial osteoblasts. The effect on mineralization was not prevented by exogenous GGPP. By inhibiting GGPPS, DGBP led to an accumulation of the GGPPS substrate FPP. We show that FPP and GGPP levels decreased during MC3T3-E1 and calvarial osteoblast differentiation, which correlated with decreased expression of HMGCR and FPP synthase. The decrease in FPP during differentiation was prevented by DGBP treatment. The accumulation of FPP following 24 h DGBP treatment correlated with activation of the glucocorticoid receptor, suggesting a potential mechanism by which DGBP-induced FPP accumulation may inhibit osteoblast differentiation. To further investigate whether FPP inhibits osteoblast differentiation, we utilized the squalene synthase (SQS) inhibitor zaragozic acid (ZGA), which causes a greater accumulation of FPP than can be achieved with GGPPS inhibition. ZGA treatment decreased osteoblast proliferation, gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization of calvarial osteoblasts. Prevention of ZGA-induced FPP accumulation with HMGCR inhibition prevented the effects of ZGA on osteoblast differentiation. Treatment of osteoblasts with exogenous FPP similarly inhibited matrix mineralization. These results suggest that the accumulation of FPP negatively regulates osteoblast differentiation. While we did not find that specific depletion of GGPP stimulates osteoblast differentiation, we obtained evidence that GGPP does negatively regulate the differentiation of these cells. Exogenous GGPP treatment inhibited primary calvarial osteoblast gene expression and matrix mineralization. Interestingly, GGPP pre-treatment increased markers of insulin signaling, despite reduced phosphorylation of the insulin receptor (InsR). Inhibition of osteoblast differentiation by GGPP led to the induction of PPARã and enhanced adipogenesis in osteoblastic cultures, suggesting that GGPP may play a role in the osteoblast versus adipocyte fate decision. Adipogenic differentiation of primary bone marrow stromal cell (BMSC) cultures was prevented by DGBP treatment. Altogether these data present novel roles for the isoprenoids FPP and GGPP in the regulation of osteoblast differentiation and have intriguing implications for the isoprenoid biosynthetic pathway in the regulation of skeletal homeostasis.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21810号 / 農博第2323号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5182(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 入江 一浩, 教授 橋本 渉, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

<p>Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH. </p><p>Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms. </p><p>The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi. </p><p> To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.</p> / Dissertation