- Email: [email protected]
W13.317 Characterization of LRP1b, a large member of the LDL receptor family and putative tumor suppressor, in the mouse
W13 W13
CELLULAR
Workshops Cellular lipid and lipoprotein transport
E. Favari, F. Bernini, F. Zimetti, S. Bertolini, G. Franceschini, L. Calabresi. REGULATION OF STEROL FLUX AT THE BLOOD-BRAIN BARRIER
U. Panzenboeck, A. Sovic, I. Kratzer, A. Hammer, E. Malle, W. Sattler.
Medical University Graz, Graz, Austria Substantial evidence has accumulated that several neut'odegenerative disorders are tightly coupled to functional lipid and lipoprotein metabolism in the periphery. Therefore, the objective of the plesent study was to investigate the involvement of key-players in reverse cholesterol/24(S)OHcholesterol tt'ansport in primat'y porcine brain capillary endothelial cells (an in vitt'o model of the BBB) and regulation of these pathways by LXR- and PPAR-agonists. We identified expression of SR-BI, ABCA1, and apoA-I by porcine brain capillat'y endothelial cells (pBCEC). Addition of cholesterol acceptors to polat'ized Transwell cultut'es resulted in substantial mobilization of cellular [3HI-cholesterol to the apical as well as basolateral compartment and was modulated by LXR and PPAR agonists. In addition, LXRand PPAR agonists regulated expression of proteins involved in reverse cholesterol transport at the apical and/or basolateral face of the membrane. In contrast to cholesterol, the majority of [3H]-24(S)OH-cholesterol was mobilized efficiently to the apical compat'tment of the in vitt'o BBB. We have shown that pBCEC ale capable of HDLtt'anscytosis and have the capacity of endogenous HDL-assembly. Since endothelial-derived lipase (EDL) is a determinant of HDL levels we have characterized the expression of EDL by pBCEC. EDL is expressed by pBCEC on mRNA and protein levels in a regulated manner. In Transwell cultures pat't of the enzyme is secreted to the apical and the basolateral compartment. Out" findings indicate that many members of the machinery that governs leverse cholesterol tt'ansport ale also expressed and active in cells that constitute the blood-brain bma'ier and could interconnect cerebral and peripheral lipid turnover.
~
73
CHOLESTEROL EFFLUX POTENTIAL OF SERA FROM SUBJECTS WITH LCAT DEFICIENCY
LIPID AND LIPOPROTEIN TRANSPORT
•
Bighetti
GENE EXPRESSION PROFILING SHOWS THAT MACROPHAGES DERIVED FROM MOUSE EMBRYONIC STEM CELLS IS AN IMPROVED IN-VITRO MODEL FOR STUDIES OF VASCULAR DISEASE
H. Lindmark, B. Rosengren, E. Hut't-Camejo, C. Bruder. AstraZeneca
R&D, M lndal. Sweden Macrophages (Mr) play an important role in the initiation and progress of the atherogenic process. They contt'ibute to the growth of atherosclerotic plaque both by affecting lipoprotein metabolism, matt'ix homeostasis, lipoprotein modification and cholesterol accumulation. Using in-vitro models of macrophages to study foam cell formation is important for understanding the mechanisms involved in the tt'ansition of the relatively simple fatty streak to a more complex type of lesion. The aim of the present work was to compat'e the expression profile of macrophages differentiated down the haematopoietic linage in mouse to peritoneal mouse macrophages and two of the most widely used mouse macrophage cell lines. Out" results showed that the embryonic stem cell derived macrophages (ES Mf) have a more similm" expression profile to peritoneal macrophages than the two mouse macrophage cell lines (J774.A1 and RAW264.7). The ES macrophages showed expression of apolipoprotein E, a key gene in atherosclerosis, which is not expressed in macrophage cell lines. Also genes involved in cholesterol efflux ale explessed in the ES Mf, whereas the J774.A1 and RAW264.7 macrophages display several alterations in the expression of these genes. In conclusion we show here that the ES Mf ale likely to provide a better in vitro model than mouse Mf cell lines to study the involvement of macrophages in atherosclerosis.
Department of Pharmacological and Biological Sciences and Applied Chemistries, University of Parma, Parma; Department of lnternal Medicine, University of Genova, Genova; Center E. Grossi Paoletti, Department of Pharmacological Sciences, University of Milan, Milan, Italy We have recently identified 9 Italian families with LCAT deficiency; genetic analysis identified 13 mutations. In homozygotes (n=9) plasma HDL-C concentration was markedly reduced (11.5-4-7.5 mg/dl) as well as plasma apoA-I (45.3-4-15.2rag/all). The analysis of HDL size showed a predominance of small HDL3 pm'ticles, with a great proportion of pre-beta HDL. Heterozygores (n=22) have slightly reduced HDL-C and apoA-I levels (39.8-t-11.4 mg/dl and 108.4-t-23.3 mg/dl), with a significant increase in pre-beta HDL. Two models of cellulaa" cholesterol efflux were used to evaluate the paa'ticipation of ABCAl-dependent and -independent pathways; 1) Fu5aH rat hepatoma cells, endogenously expressing high levels of the scavenger receptor class B type I (SR-BI) and low levels of ABCA1, and 2) J774 mut'ine macrophages expressing high levels of ABCA1 upon tt'eatment with cpt-cAMP (0.3mM, overnight) and low levels of SR-BI. In Fu5AH cells, cholesterol efflux to sera fi'om the homozygotes was significantly reduced compaaed with heterozygotes and contt'ol sera, which were not different. When the ABCAl-mediated efflux was examined, efflux potential of the different sera was in the following order:homozygotes>heterozygotes>contt'ols, and was stt'ongly related with the percentage of pre-beta HDL, suggesting that despite the dramatic hypoalphalipoproteinemia, LCAT deficient homozygotes have efficient HDL. Whether the high efficiency of these HDL for cell cholesterol uptake would result in an improved reverse cholesterol tt'ansport remains to be established.
CHARACTERIZATION OF LRPlb, A LARGE MEMBER OF THE LDL RECEPTOR FAMILY AND PUTATIVE TUMOR SUPPRESSOR, IN THE MOUSE P. Mat'schang, J. Brich, E. Weeber, J. Sweatt, J. Shelton, J. Richardson, R. Hammer, J. Herz. Department of Molecular Genetics, Department of
Internal Medicine, Department of Pathology, and Department of Biochemistry, UT Southwestern Medical Center, Dallas; Division of Neuroscience, Baylor College of Medicine, Houston, USA LRPlb and the closely related LRP1 are large members of the LDL receptor family. At the protein level LRPlb is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cat'go tt'ansport and cellulat" signaling. Somatic LRPlb mutations fi'equently occur" in non-small cell lung cancer and ut'othelial cancers, suggesting a role as a tumor suppressor. We studied the expression of LRPlb in the mouse by RT-PCR and by Western blotting using a rabbit antibody raised against the cat'boxyl-terminus of the receptor. LRPlb expression is largely confined to the brain, where a full-length form and an alternatively spliced receptor lacking exon 90 ale present. The alternatively spliced form is also expressed in the adrenal gland and in the testis. Despite the presence of a potential fut'in cleavage site and in contt'ast to LRP1, immunoblotting for LRPlb reveals the presence of a single 600-kDa polypeptide species in brain membranes as well as in 293 cells tt'ansfected with an LRPlb minfl'eceptor containing the 4th ligand binding domain region. Using a yeast two-hybrid approach, we have identified two intt'acellulat" proteins, the postsynaptic density protein95 and the aryl hydrocarbon receptolqnteracting protein, that bind to the intt'acellular domain of LRPlb. In addition, we have found several potential ligands that bind to the extt'acellulat" domain by affinity purification. Using homologous DNA recombination, homozygous LRPlb knockout mice were generated. These LRPlb-/- mice appeaL"normal, probably due to functional redundancy of other receptors, most likely LRP1. These and fut'ther studies may help to understand the physiological role of LRPlb as well as the role of LRPlb in cancer development.
~
LIPEMIC AND GLYCEMIC RESPONSES TO CIPROFIBRATE TREATMENT IN GENETIC DYSLIPIDEMIC MICE
E.J.B. Bighetti, M.E.C. Amaral, J.A. Berti, A.C. Casquero, A.C. Boschero, H.C.F. Olivefl'a. Instituto de Biologia, Universidade Estadual de Campinas,
Campinas, Brazil The aim of the plesent study was to investigate the effects of the PPARc~
74th EAS Congress, 17-20 April 2004, Seville, Spain
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iiiiii:".O.'iiiiiiiiiiiiiiii
iiiii iiiiiiiiiiiiiii iiiiii:O.'iiiiiiiiiiiiiiii
CELLULAR
Workshops Cellular lipid and lipoprotein transport
E. Favari, F. Bernini, F. Zimetti, S. Bertolini, G. Franceschini, L. Calabresi. REGULATION OF STEROL FLUX AT THE BLOOD-BRAIN BARRIER
U. Panzenboeck, A. Sovic, I. Kratzer, A. Hammer, E. Malle, W. Sattler.
Medical University Graz, Graz, Austria Substantial evidence has accumulated that several neut'odegenerative disorders are tightly coupled to functional lipid and lipoprotein metabolism in the periphery. Therefore, the objective of the plesent study was to investigate the involvement of key-players in reverse cholesterol/24(S)OHcholesterol tt'ansport in primat'y porcine brain capillary endothelial cells (an in vitt'o model of the BBB) and regulation of these pathways by LXR- and PPAR-agonists. We identified expression of SR-BI, ABCA1, and apoA-I by porcine brain capillat'y endothelial cells (pBCEC). Addition of cholesterol acceptors to polat'ized Transwell cultut'es resulted in substantial mobilization of cellular [3HI-cholesterol to the apical as well as basolateral compartment and was modulated by LXR and PPAR agonists. In addition, LXRand PPAR agonists regulated expression of proteins involved in reverse cholesterol transport at the apical and/or basolateral face of the membrane. In contrast to cholesterol, the majority of [3H]-24(S)OH-cholesterol was mobilized efficiently to the apical compat'tment of the in vitt'o BBB. We have shown that pBCEC ale capable of HDLtt'anscytosis and have the capacity of endogenous HDL-assembly. Since endothelial-derived lipase (EDL) is a determinant of HDL levels we have characterized the expression of EDL by pBCEC. EDL is expressed by pBCEC on mRNA and protein levels in a regulated manner. In Transwell cultures pat't of the enzyme is secreted to the apical and the basolateral compartment. Out" findings indicate that many members of the machinery that governs leverse cholesterol tt'ansport ale also expressed and active in cells that constitute the blood-brain bma'ier and could interconnect cerebral and peripheral lipid turnover.
~
73
CHOLESTEROL EFFLUX POTENTIAL OF SERA FROM SUBJECTS WITH LCAT DEFICIENCY
LIPID AND LIPOPROTEIN TRANSPORT
•
Bighetti
GENE EXPRESSION PROFILING SHOWS THAT MACROPHAGES DERIVED FROM MOUSE EMBRYONIC STEM CELLS IS AN IMPROVED IN-VITRO MODEL FOR STUDIES OF VASCULAR DISEASE
H. Lindmark, B. Rosengren, E. Hut't-Camejo, C. Bruder. AstraZeneca
R&D, M lndal. Sweden Macrophages (Mr) play an important role in the initiation and progress of the atherogenic process. They contt'ibute to the growth of atherosclerotic plaque both by affecting lipoprotein metabolism, matt'ix homeostasis, lipoprotein modification and cholesterol accumulation. Using in-vitro models of macrophages to study foam cell formation is important for understanding the mechanisms involved in the tt'ansition of the relatively simple fatty streak to a more complex type of lesion. The aim of the present work was to compat'e the expression profile of macrophages differentiated down the haematopoietic linage in mouse to peritoneal mouse macrophages and two of the most widely used mouse macrophage cell lines. Out" results showed that the embryonic stem cell derived macrophages (ES Mf) have a more similm" expression profile to peritoneal macrophages than the two mouse macrophage cell lines (J774.A1 and RAW264.7). The ES macrophages showed expression of apolipoprotein E, a key gene in atherosclerosis, which is not expressed in macrophage cell lines. Also genes involved in cholesterol efflux ale explessed in the ES Mf, whereas the J774.A1 and RAW264.7 macrophages display several alterations in the expression of these genes. In conclusion we show here that the ES Mf ale likely to provide a better in vitro model than mouse Mf cell lines to study the involvement of macrophages in atherosclerosis.
Department of Pharmacological and Biological Sciences and Applied Chemistries, University of Parma, Parma; Department of lnternal Medicine, University of Genova, Genova; Center E. Grossi Paoletti, Department of Pharmacological Sciences, University of Milan, Milan, Italy We have recently identified 9 Italian families with LCAT deficiency; genetic analysis identified 13 mutations. In homozygotes (n=9) plasma HDL-C concentration was markedly reduced (11.5-4-7.5 mg/dl) as well as plasma apoA-I (45.3-4-15.2rag/all). The analysis of HDL size showed a predominance of small HDL3 pm'ticles, with a great proportion of pre-beta HDL. Heterozygores (n=22) have slightly reduced HDL-C and apoA-I levels (39.8-t-11.4 mg/dl and 108.4-t-23.3 mg/dl), with a significant increase in pre-beta HDL. Two models of cellulaa" cholesterol efflux were used to evaluate the paa'ticipation of ABCAl-dependent and -independent pathways; 1) Fu5aH rat hepatoma cells, endogenously expressing high levels of the scavenger receptor class B type I (SR-BI) and low levels of ABCA1, and 2) J774 mut'ine macrophages expressing high levels of ABCA1 upon tt'eatment with cpt-cAMP (0.3mM, overnight) and low levels of SR-BI. In Fu5AH cells, cholesterol efflux to sera fi'om the homozygotes was significantly reduced compaaed with heterozygotes and contt'ol sera, which were not different. When the ABCAl-mediated efflux was examined, efflux potential of the different sera was in the following order:homozygotes>heterozygotes>contt'ols, and was stt'ongly related with the percentage of pre-beta HDL, suggesting that despite the dramatic hypoalphalipoproteinemia, LCAT deficient homozygotes have efficient HDL. Whether the high efficiency of these HDL for cell cholesterol uptake would result in an improved reverse cholesterol tt'ansport remains to be established.
CHARACTERIZATION OF LRPlb, A LARGE MEMBER OF THE LDL RECEPTOR FAMILY AND PUTATIVE TUMOR SUPPRESSOR, IN THE MOUSE P. Mat'schang, J. Brich, E. Weeber, J. Sweatt, J. Shelton, J. Richardson, R. Hammer, J. Herz. Department of Molecular Genetics, Department of
Internal Medicine, Department of Pathology, and Department of Biochemistry, UT Southwestern Medical Center, Dallas; Division of Neuroscience, Baylor College of Medicine, Houston, USA LRPlb and the closely related LRP1 are large members of the LDL receptor family. At the protein level LRPlb is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cat'go tt'ansport and cellulat" signaling. Somatic LRPlb mutations fi'equently occur" in non-small cell lung cancer and ut'othelial cancers, suggesting a role as a tumor suppressor. We studied the expression of LRPlb in the mouse by RT-PCR and by Western blotting using a rabbit antibody raised against the cat'boxyl-terminus of the receptor. LRPlb expression is largely confined to the brain, where a full-length form and an alternatively spliced receptor lacking exon 90 ale present. The alternatively spliced form is also expressed in the adrenal gland and in the testis. Despite the presence of a potential fut'in cleavage site and in contt'ast to LRP1, immunoblotting for LRPlb reveals the presence of a single 600-kDa polypeptide species in brain membranes as well as in 293 cells tt'ansfected with an LRPlb minfl'eceptor containing the 4th ligand binding domain region. Using a yeast two-hybrid approach, we have identified two intt'acellulat" proteins, the postsynaptic density protein95 and the aryl hydrocarbon receptolqnteracting protein, that bind to the intt'acellular domain of LRPlb. In addition, we have found several potential ligands that bind to the extt'acellulat" domain by affinity purification. Using homologous DNA recombination, homozygous LRPlb knockout mice were generated. These LRPlb-/- mice appeaL"normal, probably due to functional redundancy of other receptors, most likely LRP1. These and fut'ther studies may help to understand the physiological role of LRPlb as well as the role of LRPlb in cancer development.
~
LIPEMIC AND GLYCEMIC RESPONSES TO CIPROFIBRATE TREATMENT IN GENETIC DYSLIPIDEMIC MICE
E.J.B. Bighetti, M.E.C. Amaral, J.A. Berti, A.C. Casquero, A.C. Boschero, H.C.F. Olivefl'a. Instituto de Biologia, Universidade Estadual de Campinas,
Campinas, Brazil The aim of the plesent study was to investigate the effects of the PPARc~
74th EAS Congress, 17-20 April 2004, Seville, Spain
iiiiiiiiiiiiiiiiiiii
iiiiii:".O.'iiiiiiiiiiiiiiii
iiiii iiiiiiiiiiiiiii iiiiii:O.'iiiiiiiiiiiiiiii