Schweinfurthins are intriguing natural product chemotherapeutics due to their potent yet selective activity and their unknown mechanism of growth inhibition in cancer. Much progress has been made in characterizing the intracellular effects of the schweinfurthins since they were first isolated from Macaranga schweinfurthii in 1986. Here, the L-type calcium channel and P- glycoprotein (Pgp) inhibitor verapamil has been found to enhance schweinfurthin- induced growth inhibition. Verapamil induces an increase in the intracellular concentration of a fluorescent schweinfurthin. However, the synergistic relationship between the schweinfurthins and verapamil is complex and not obvious in that verapamil fails to increase the intracellular concentration of a schweinfurthin analogue that is a known substrate of Pgp. Schweinfurthins are also found to induce alterations to cholesterol homeostasis by increasing the expression of the cholesterol efflux pump ABCA1 in an apparent liver X receptor- independent fashion. In addition, schweinfurthin treatment blunts epidermal growth factor downstream activation and phosphorylation of Akt. Lastly, a schweinfurthin-resistant cell line has been created and characterized for resistance to schweinfurthin-induced growth inhibition. The variety of intracellular effects characteristic of schweinfurthin treatment described here provide mechanistic framework for identifying the potential target and mechanism of growth inhibition for the schweinfurthins.

ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21815号 / 農博第2328号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5187(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 矢﨑 一史, 教授 植田 充美 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM