Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, which is generated by activation of sp... more Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, which is generated by activation of sphingosine kinase (SK) 1 and/or 2 in most mammalian cells with various stimuli, including the oral pathogen Aggregatibacter actinomycetemcomitans. S1P signaling has been shown to regulate the migration of monocytes and macrophages (osteoclast precursors) from the circulation to bone tissues and affect bone homeostasis. We aimed to determine the effects of SK1 deficiency on S1P generation, proinflammatory cytokine production, chemotaxis of monocytes and macrophages, and periodontitis induced by A. actinomycetemcomitans. Murine bone marrow-derived monocytes and macrophages (BMMs) from SK1 knockout (KO) mice or wild-type (WT) mice were either untreated or exposed to A. actinomycetemcomitans. The mRNA levels of SK1, SK2 and intracellular sphingolipid levels were quantified. In addition, murine WT BMMs were treated with vehicle, S1P, with or without A. actinomycetemcomitans and the mRNA levels...
The very low density lipoprotein (VLDL) receptor is closely related in structure to the low densi... more The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the...
Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of v... more Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of vasculogenesis in the mouse embryo. At stages preceding the formation of blood vessels (7.5-8 dpc) in the embryo proper, yolk sac, and allantois, the S1P receptor S1P(2) is expressed in conjunction with S1P(1) and/or S1P(3). Additionally, sphingosine kinase-2 (SK2), an enzyme that catalyzes the formation of S1P, is expressed in these tissues throughout periods of vasculogenesis. Using the cultured mouse allantois explant model of blood vessel formation, we found that vasculogenesis was dependent on S1P signaling. We showed that S1P could replace the ability of serum to promote vasculogenesis in cultured allantois explants. Instead of small poorly reticulated clusters of rounded endothelial cells that formed under serum-free conditions, S1P promoted the formation of elongated endothelial cells that arranged into expansive branched networks of capillary-like vessels. These effects could not be reproduced by vascular endothelial growth factor or basic fibroblast growth factor administration. The ability of S1P to promote blood vessel formation was not due to effects on cell survival or on changes in numbers of endothelial cells (Flk1(+)/PECAM(+)), angioblasts (Flk1(+)/PECAM(-)), or undifferentiated mesodermal cells (Flk1(-)/PECAM(-)). The S1P effect on blood vessel formation was attributed to it promoting migratory activities of angioblasts and early endothelial cells required for the expansion of vascular networks. Together, our findings suggest that migratory events critical to the de novo formation of blood vessels are under the influence of S1P, possibly synthesized via the action of SK2, with signaling mediated by S1P receptors that include S1P(1), S1P(2), and S1P(3).
The very low density lipoprotein (VLDL) receptor binds apolipoprotein E-rich lipoproteins as well... more The very low density lipoprotein (VLDL) receptor binds apolipoprotein E-rich lipoproteins as well as the 39-kDa receptor-associated protein (RAP). Ligand blotting experiments using RAP and immunoblotting experiments using an anti-VLDL receptor IgG detected the VLDL receptor in detergent extracts of human aortic endothelial cells, human umbilical vein endothelial cells, and human aortic smooth muscle cells. To gain insight into the role of the VLDL receptor in the vascular endothelium, its ligand binding properties were further characterized. In vitro binding experiments documented that lipoprotein lipase (LpL), a key enzyme in lipoprotein catabolism, binds with high affinity to purified VLDL receptor. In addition, urokinase complexed with plasminogen activator-inhibitor type I (uPA.PAI-1) also bound to the purified VLDL receptor with high affinity. To assess the capacity of the VLDL receptor to mediate the cellular internalization of ligands, an adenoviral vector was used to introduce the VLDL receptor gene into a murine embryonic fibroblast cell line deficient in the VLDL receptor and the LDL receptor-related protein, another endocytic receptor known to bind LpL and uPA.PAI-1 complexes. Infected fibroblasts that express the VLDL receptor mediate the cellular internalization of 125I-labeled LpL and uPA.PAI-1 complexes, leading to their degradation. Non-infected fibroblasts or fibroblasts infected with the lacZ gene did not internalize these ligands. These studies confirm that the VLDL receptor binds to and mediates the catabolism of LpL and uPA.PAI-1 complexes. Thus, the VLDL receptor may play a unique role on the vascular endothelium in lipoprotein catabolism by regulating levels of LpL and in the regulation of fibrinolysis by facilitating the removal of urokinase complexed with its inhibitor.
Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a div... more Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a diverse role in biological processes. Newly synthesized TSP-1 either is incorporated into the matrix or binds to the cell surface where it is rapidly internalized and degraded. TSP-1 catabolism is mediated by the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that is a member of the low density lipoprotein receptor family. Using adenovirus-mediated gene transfer experiments, we demonstrate that the very low density lipoprotein receptor can also bind and internalize TSP-1. An objective of the current investigation was to identify the portion of TSP-1 that binds to these endocytic receptors. The current studies found that the amino-terminal heparin binding domain (HBD, residues 1-214) of mouse TSP-1, when prepared as a fusion protein with glutathione S-transferase (GST), bound to purified LRP with an apparent KD ranging from 10 to 25 nM. Recombinant HBD (rHBD) purified following proteolytic cleavage of GST-HBD, also bound to purified LRP, but with an apparent KD of 830 nM. The difference in affinity was attributed to the fact that GST-HBD exists in solution as a dimer, whereas rHBD is a monomer. Like TSP-1, 125I-labeled GST-HBD or 125I-labeled rHBD were internalized and degraded by wild type fibroblasts that express LRP, but not by fibroblasts that are genetically deficient in LRP. The catabolism of both 125I-labeled GST-HBD and rHBD in wild type fibroblast was blocked by the 39-kDa receptor-associated protein, an inhibitor of LRP function. GST-HBD and rHBD both completely blocked catabolism of 125I-labeled TSP-1 in a dose-dependent manner, as did antibodies prepared against the HBD. Taken together, these data provide compelling evidence that the amino-terminal domain of TSP-1 binds to LRP and thus the recognition determinants on TSP-1 for both LRP and for cell surface proteoglycans reside within the same TSP-1 domain. Further, high affinity binding of TSP-1 to LRP likely results from the trimeric structure of TSP-1.
High density lipoproteins (HDL) are major plasma carriers of sphingosine 1-phosphate (S1P). Here ... more High density lipoproteins (HDL) are major plasma carriers of sphingosine 1-phosphate (S1P). Here we show that HDL increases endothelial barrier integrity as measured by electric cell substrate impedance sensing. S1P was implicated as the mediator in this process through findings showing that pertussis toxin, an inhibitor of Gi-coupled S1P receptors, as well as antagonists of the S1P receptor, S1P1, inhibited barrier enhancement by HDL. Additional findings show that HDL stimulates endothelial cell activation of Erk1/2 and Akt, signaling pathway intermediates that have been implicated in S1P-dependent endothelial barrier activity. HDL was also found to promote endothelial cell motility, a process that may also relate to endothelial barrier function in the context of a vascular injury response. The effects of HDL on endothelial cell Erk1/2 and Akt activation and motility were suppressed by pertussis toxin and S1P1 antagonists. However, both HDL-induced barrier enhancement and HDL-induced motility showed a greater dependence on Akt activation as compared with Erk1/2 activation. Together, the findings indicate that HDL has endothelial barrier promoting activities, which are attributable to its S1P component and signaling through the S1P1/Akt pathway.
The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds sev... more The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds several ligands including the activated form of the pan-proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*) and amyloid precursor protein, two ligands genetically linked to Alzheimer's disease. To delineate the contribution of LRP to this disease, it will be necessary to identify the sites on this receptor which are responsible for recognizing these and other ligands to assist in the development of specific inhibitors. Structurally, LRP contains four clusters of cysteine-rich repeats, yet studies thus far suggest that only two of these clusters (clusters II and IV) bind ligands. Identifying binding sites within LRP for certain ligands, such as alpha(2)M*, has proven to be difficult. To accomplish this, we mapped the binding site on LRP for two inhibitors of alpha(2)M* uptake, monoclonal antibody 8G1 and an amino-terminal fragment of receptor-associated protein (RAP D1D2). Surprisingly, the inhibitors recognized different clusters of ligand binding repeats: 8G1 bound to repeats within cluster I, whereas the RAP fragment bound to repeats within cluster II. A recombinant LRP mini-receptor containing the repeats from cluster I along with three ligand binding repeats from cluster II was effective in mediating the internalization of (125)I-labeled alpha(2)M*. Together, these studies indicate that ligand binding repeats from both cluster I and II cooperate to generate a high affinity binding site for alpha(2)M*, and they suggest a strategy for developing specific inhibitors to block alpha(2)M* binding to LRP by identifying molecules capable of binding repeats in cluster I.
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, which is generated by activation of sp... more Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, which is generated by activation of sphingosine kinase (SK) 1 and/or 2 in most mammalian cells with various stimuli, including the oral pathogen Aggregatibacter actinomycetemcomitans. S1P signaling has been shown to regulate the migration of monocytes and macrophages (osteoclast precursors) from the circulation to bone tissues and affect bone homeostasis. We aimed to determine the effects of SK1 deficiency on S1P generation, proinflammatory cytokine production, chemotaxis of monocytes and macrophages, and periodontitis induced by A. actinomycetemcomitans. Murine bone marrow-derived monocytes and macrophages (BMMs) from SK1 knockout (KO) mice or wild-type (WT) mice were either untreated or exposed to A. actinomycetemcomitans. The mRNA levels of SK1, SK2 and intracellular sphingolipid levels were quantified. In addition, murine WT BMMs were treated with vehicle, S1P, with or without A. actinomycetemcomitans and the mRNA levels...
The very low density lipoprotein (VLDL) receptor is closely related in structure to the low densi... more The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the...
Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of v... more Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of vasculogenesis in the mouse embryo. At stages preceding the formation of blood vessels (7.5-8 dpc) in the embryo proper, yolk sac, and allantois, the S1P receptor S1P(2) is expressed in conjunction with S1P(1) and/or S1P(3). Additionally, sphingosine kinase-2 (SK2), an enzyme that catalyzes the formation of S1P, is expressed in these tissues throughout periods of vasculogenesis. Using the cultured mouse allantois explant model of blood vessel formation, we found that vasculogenesis was dependent on S1P signaling. We showed that S1P could replace the ability of serum to promote vasculogenesis in cultured allantois explants. Instead of small poorly reticulated clusters of rounded endothelial cells that formed under serum-free conditions, S1P promoted the formation of elongated endothelial cells that arranged into expansive branched networks of capillary-like vessels. These effects could not be reproduced by vascular endothelial growth factor or basic fibroblast growth factor administration. The ability of S1P to promote blood vessel formation was not due to effects on cell survival or on changes in numbers of endothelial cells (Flk1(+)/PECAM(+)), angioblasts (Flk1(+)/PECAM(-)), or undifferentiated mesodermal cells (Flk1(-)/PECAM(-)). The S1P effect on blood vessel formation was attributed to it promoting migratory activities of angioblasts and early endothelial cells required for the expansion of vascular networks. Together, our findings suggest that migratory events critical to the de novo formation of blood vessels are under the influence of S1P, possibly synthesized via the action of SK2, with signaling mediated by S1P receptors that include S1P(1), S1P(2), and S1P(3).
The very low density lipoprotein (VLDL) receptor binds apolipoprotein E-rich lipoproteins as well... more The very low density lipoprotein (VLDL) receptor binds apolipoprotein E-rich lipoproteins as well as the 39-kDa receptor-associated protein (RAP). Ligand blotting experiments using RAP and immunoblotting experiments using an anti-VLDL receptor IgG detected the VLDL receptor in detergent extracts of human aortic endothelial cells, human umbilical vein endothelial cells, and human aortic smooth muscle cells. To gain insight into the role of the VLDL receptor in the vascular endothelium, its ligand binding properties were further characterized. In vitro binding experiments documented that lipoprotein lipase (LpL), a key enzyme in lipoprotein catabolism, binds with high affinity to purified VLDL receptor. In addition, urokinase complexed with plasminogen activator-inhibitor type I (uPA.PAI-1) also bound to the purified VLDL receptor with high affinity. To assess the capacity of the VLDL receptor to mediate the cellular internalization of ligands, an adenoviral vector was used to introduce the VLDL receptor gene into a murine embryonic fibroblast cell line deficient in the VLDL receptor and the LDL receptor-related protein, another endocytic receptor known to bind LpL and uPA.PAI-1 complexes. Infected fibroblasts that express the VLDL receptor mediate the cellular internalization of 125I-labeled LpL and uPA.PAI-1 complexes, leading to their degradation. Non-infected fibroblasts or fibroblasts infected with the lacZ gene did not internalize these ligands. These studies confirm that the VLDL receptor binds to and mediates the catabolism of LpL and uPA.PAI-1 complexes. Thus, the VLDL receptor may play a unique role on the vascular endothelium in lipoprotein catabolism by regulating levels of LpL and in the regulation of fibrinolysis by facilitating the removal of urokinase complexed with its inhibitor.
Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a div... more Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a diverse role in biological processes. Newly synthesized TSP-1 either is incorporated into the matrix or binds to the cell surface where it is rapidly internalized and degraded. TSP-1 catabolism is mediated by the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that is a member of the low density lipoprotein receptor family. Using adenovirus-mediated gene transfer experiments, we demonstrate that the very low density lipoprotein receptor can also bind and internalize TSP-1. An objective of the current investigation was to identify the portion of TSP-1 that binds to these endocytic receptors. The current studies found that the amino-terminal heparin binding domain (HBD, residues 1-214) of mouse TSP-1, when prepared as a fusion protein with glutathione S-transferase (GST), bound to purified LRP with an apparent KD ranging from 10 to 25 nM. Recombinant HBD (rHBD) purified following proteolytic cleavage of GST-HBD, also bound to purified LRP, but with an apparent KD of 830 nM. The difference in affinity was attributed to the fact that GST-HBD exists in solution as a dimer, whereas rHBD is a monomer. Like TSP-1, 125I-labeled GST-HBD or 125I-labeled rHBD were internalized and degraded by wild type fibroblasts that express LRP, but not by fibroblasts that are genetically deficient in LRP. The catabolism of both 125I-labeled GST-HBD and rHBD in wild type fibroblast was blocked by the 39-kDa receptor-associated protein, an inhibitor of LRP function. GST-HBD and rHBD both completely blocked catabolism of 125I-labeled TSP-1 in a dose-dependent manner, as did antibodies prepared against the HBD. Taken together, these data provide compelling evidence that the amino-terminal domain of TSP-1 binds to LRP and thus the recognition determinants on TSP-1 for both LRP and for cell surface proteoglycans reside within the same TSP-1 domain. Further, high affinity binding of TSP-1 to LRP likely results from the trimeric structure of TSP-1.
High density lipoproteins (HDL) are major plasma carriers of sphingosine 1-phosphate (S1P). Here ... more High density lipoproteins (HDL) are major plasma carriers of sphingosine 1-phosphate (S1P). Here we show that HDL increases endothelial barrier integrity as measured by electric cell substrate impedance sensing. S1P was implicated as the mediator in this process through findings showing that pertussis toxin, an inhibitor of Gi-coupled S1P receptors, as well as antagonists of the S1P receptor, S1P1, inhibited barrier enhancement by HDL. Additional findings show that HDL stimulates endothelial cell activation of Erk1/2 and Akt, signaling pathway intermediates that have been implicated in S1P-dependent endothelial barrier activity. HDL was also found to promote endothelial cell motility, a process that may also relate to endothelial barrier function in the context of a vascular injury response. The effects of HDL on endothelial cell Erk1/2 and Akt activation and motility were suppressed by pertussis toxin and S1P1 antagonists. However, both HDL-induced barrier enhancement and HDL-induced motility showed a greater dependence on Akt activation as compared with Erk1/2 activation. Together, the findings indicate that HDL has endothelial barrier promoting activities, which are attributable to its S1P component and signaling through the S1P1/Akt pathway.
The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds sev... more The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds several ligands including the activated form of the pan-proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*) and amyloid precursor protein, two ligands genetically linked to Alzheimer's disease. To delineate the contribution of LRP to this disease, it will be necessary to identify the sites on this receptor which are responsible for recognizing these and other ligands to assist in the development of specific inhibitors. Structurally, LRP contains four clusters of cysteine-rich repeats, yet studies thus far suggest that only two of these clusters (clusters II and IV) bind ligands. Identifying binding sites within LRP for certain ligands, such as alpha(2)M*, has proven to be difficult. To accomplish this, we mapped the binding site on LRP for two inhibitors of alpha(2)M* uptake, monoclonal antibody 8G1 and an amino-terminal fragment of receptor-associated protein (RAP D1D2). Surprisingly, the inhibitors recognized different clusters of ligand binding repeats: 8G1 bound to repeats within cluster I, whereas the RAP fragment bound to repeats within cluster II. A recombinant LRP mini-receptor containing the repeats from cluster I along with three ligand binding repeats from cluster II was effective in mediating the internalization of (125)I-labeled alpha(2)M*. Together, these studies indicate that ligand binding repeats from both cluster I and II cooperate to generate a high affinity binding site for alpha(2)M*, and they suggest a strategy for developing specific inhibitors to block alpha(2)M* binding to LRP by identifying molecules capable of binding repeats in cluster I.
Uploads
Papers by K. Argraves