U18666A, a cholesterol-inhibition agent, modulates human neuronal nicotinic acetylcholine receptors heterologously expressed in SH-EP1 cell line

In this study, we evaluate the effects of (3β)‐3‐[2‐(diethylamino)ethoxy]androst‐5‐en‐17‐one dihydrochloride (U18666A), a cholesterol synthesis/transporter inhibitor, on selected human neuronal nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the SH‐EP1 cell line using whole‐cell patch‐clamp recordings. The results indicate that with 2‐min pretreatment, U18666A inhibited different nAChR subtypes with a rank‐order of potency (IC₅₀ of whole‐cell peak current): α4β2 (8.0 ± 3.0 nM) > α3β2 (1.7 ± 0.4 μM) > α4β4 (26 ± 7.2 μM) > α7 (> 100 μM), suggesting this compound is more selective to α4β2‐nAChRs. Thus, the pharmacological profiles and mechanisms of U18666A acting on α4β2‐nAChRs were investigated in detail. U18666A suppresses both peak and steady state components of whole‐cell currents mediated by human α4β2‐nAChRs in response to nicotine. In nicotine‐induced concentration–response curves, U18666A reduces nicotine‐induced current at maximally effective agonist concentrations without influencing nicotine’s EC₅₀ value, suggesting a non‐competitive inhibition. U18666A‐induced inhibition of nAChR function is concentration‐, voltage‐, and use‐dependent, suggesting an open channel block. Taken into consideration of ～10 000‐fold enhancement of the potency of U18666A after 2‐min pre‐treatment, this compound also likely inhibits α4β2‐nAChRs through a close channel block. In addition, the U18666A‐induced inhibition in α4β2‐nAChRs is not mediated by either increased receptor endocytosis or altered cell cholesterol. These data indicate that U18666A is a potent antagonist of α4β2‐nAChRs and may be useful as a tool in the functional characterization and pharmacological profiling of nAChRs, as well as a potential candidate for smoking cessation.     

U18666A Inhibits Intracellular Cholesterol Transport and Neurotransmitter Release in Human Neuroblastoma Cells

To determine if neurochemical function might be impaired in cell models with altered cholesterol balance, we studied the effects of U18666A (3--[(2-diethyl-amino)ethoxy]androst-5-en-17-one) on intracellular cholesterol metabolism in three human neuroblastoma cell lines (SK-N-SH, SK-N-MC, and SH-SY5Y). U18666A (0.2 g/ml) completely inhibited low density lipoprotein (LDL)-stimulated cholesterol esterification in SK-N-SH cells, while cholesterol esterification stimulated by 25-hydroxycholesterol or bacterial sphingomyelinase was unaffected or partially inhibited, respectively. U18666A also blocked LDL-stimulated downregulation of LDL receptor and caused lysosomal accumulation of cholesterol as measured by filipin staining. U18666A treatment for 18 h resulted in 70% inhibition of K⁺-evoked norepinephrine release in phorbol esterdifferentiated SH-SY5Y cells, while release stimulated by the calcium ionophore A23187 was only slightly affected. These results suggest that U18666A may preferentially block a voltage-regulated Ca²⁺ channel involved in norepinephrine release and that alterations in neurotransmitter secretion might be a feature of disorders such as Niemann-Pick Type C, in which intracellular cholesterol transport and distribution are impaired.     

Accumulation of tyrosinase in the endolysosomal compartment is induced by U18666A

The 3beta-(2-diethylaminoethoxy)-androstenone HCl (U18666A), progesterone and several cationic amphiphilic drugs have been shown to alter the trafficking of a number of intracellular membrane proteins including CD63/Lamp-3, insulin growth factor 2/mannose 6-phosphate receptor (IGF2/MPR), and the Niemann-Pick C1 gene product (NPC1) as well as ganglioside GM1. We have examined the effects of these compounds on cultured melanocytes at concentrations that have been shown to effectively alter intracellular trafficking. Treatment of melanocytes with U18666A (2.5 micro M) or progesterone (15 micro M) for 96 h decreased melanin content an average of 67% as compared with control without lowering the total cellular tyrosinase activity. Steroidal alkaloids that preferentially act on the Sonic Hedgehog signaling pathway showed no related specificity in their ability to decrease pigmentation. In melanocytes treated with U18666A, tyrosinase accumulates in a compartment that contains both lysosome-associated membrane protein-1 (Lamp 1) and MPR, and stains with filipin, consistent with cholesterol-laden late endosomes/lysosomes. Our results suggest that tyrosinase, like the NPC1 gene product, traverses a U18666A-sensitive trafficking pathway.     

Bone Morphogenetic Proteins Signal Via SMAD and Mitogen-activated Protein (MAP) Kinase Pathways at Distinct Times during Osteoclastogenesis

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII^fl/fl;lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.     

The mechanism of the effect of U18666a on blocking the activity of 3β-hydroxysterol Δ-24-reductase (DHCR24): molecular dynamics simulation study and free energy analysis

DHCR24 encodes 3β-hydroxysterol-Δ²⁴-reductase (DHCR24) catalyzing the cholesterol synthesis from desmosterol using the flavin adenine dinucleotide (FAD) as a co-factor. It is generally accepted that U18666a inhibits the reductase activity of DHCR24, but the detailed mechanism remains elusive. To explore the mechanism of the inhibitory effect of U18666a on DHCR24, we performed molecular dynamics (MD) simulations of two complexes including complexes of DHCR24-FAD-desmosterol enzymatic reactive components with and without the inhibitor U18666a. We found that the U18666a bound into the hydrophobic package near the FAD package of DHCR24. Furthermore, binding free energy of DHCR24 and desmosterol without U18666a is ?54.86 kcal/mol, while the system with U18666a is ?62.23 kcal/mol, suggesting that the affinity of the substrate desmosterol to DHCR24 was increased in response to the U18666a. In addition, U18666a interacts with FAD by newly forming three hydrogen bonds with Lys292, Lys367, and Gly438 of DHCR24. Finally, secondary structural analysis data obtained from the surrounding hot spots showed that U18666a induced dramatic secondary structural changes around the key residues in the interaction of DHCR24, FAD, and desmosterol. Taken together, these results for the first time demonstrate at the molecular structure level that U18666a blocks DHCR24 activity through an allosteric inhibiting mechanism, which may provide new insight into the development of a new type of cholesterol-lowering drug targeting to block the activity of DHCR24.     

Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells

Periodontal ligament (PDL) cells are mechanosensitive and have the potential to differentiate into osteoblast-like cells under the influence of cyclic tensile force (CTF). CTF modulates the expression of regulatory proteins including bone morphogenetic proteins (BMPs), which are essential for the homeostasis of the periodontium. Among the BMPs, BMP9 is one of the most potent osteogenic BMPs. It is yet unknown whether CTF affects the expression of BMP9 and mineralization. Here, we demonstrated that continuously applied CTF for only the first 6 hr stimulated the synthesis of BMP9 and induced mineral deposition within 14 days by human PDL cells. Stimulation of BMP9 expression depended on ATP and P2Y ₁ receptors. Apyrase, an ecto-ATPase, inhibited CTF-mediated ATP-induced BMP9 expression. The addition of ATP increased the expression of BMP9. Loss of function experiments using suramin (a broad-spectrum P2Y antagonist), MRS2179 (a specific P2Y ₁ receptor antagonist), MRS 2365 (a specific P2Y ₁ agonist), U-73122 (a phospholipase C [PLC] inhibitor), and thapsigargin (enhancer of intracytosolic calcium) revealed the participation of P2Y ₁ in regulating the expression of BMP9. This was mediated by an increased level of intracellular Ca ²⁺ through the PLC pathway. A neutralizing anti-BMP9 antibody decreased mineral deposition, which was stimulated by CTF for almost 45% indicating a role of BMP9 in an in vitro mineralization. Collectively, our findings suggest an essential modulatory role of CTF in the homeostasis and regeneration of the periodontium.     

Characterization of Chinese hamster ovary cells that are resistant to 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibition of low density lipoprotein-derived cholesterol metabolism

The pharmacological agent U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibits the intracellular transport of low density lipoprotein (LDL)-derived cholesterol in Chinese hamster ovary (CHO) cells. LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated cells causing delayed LDL-mediated regulation of cellular cholesterol metabolism and impaired movement of LDL-derived cholesterol to other cell membranes. As a result of impaired LDL-derived cholesterol transport, LDL-dependent growth of CHO cells is also inhibited by U18666A. By selecting for cell growth in the presence of U18666A, we have identified a CHO cell line, designated U18R, that is resistant to U18666A-inhibition of LDL-derived cholesterol trafficking. When compared to parental CHO cells, U18R cells are relatively resistant to U18666A inhibition of LDL-derived cholesterol transport as well as LDL-mediated regulation of cellular cholesterol metabolism. In cell fusion experiments, the U18666A resistance observed in U18R cells displays a dominant phenotype. Identification of the U18666A-resistant factor may provide important insights toward the understanding of intracellular LDL-derived cholesterol regulation and trafficking.     

Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.     

NPC1-containing compartment of human granulosa-lutein cells: a role in the intracellular trafficking of cholesterol supporting steroidogenesis

Steroidogenic cells represent unique systems for the exploration of intracellular cholesterol trafficking. We employed cytochemical and biochemical methods to explore the expression, regulation, and function of the Niemann-Pick C1 protein (NPC1) in human granulosa-lutein cells. NPC1 was localized in a subset of lysosome-associated membrane glycoprotein 2 (LAMP-2)-positive vesicles. By analyzing the sensitivity of NPC1 N-linked oligosaccharide chains to glycosidases and neuraminidase, evidence was obtained for movement of nascent NPC1 from the endoplasmic reticulum through the medial and trans compartments of the Golgi apparatus prior to its appearance in cytoplasmic vesicles. NPC1 protein content and the morphology and cellular distribution of NPC1-containing vesicles were not affected by treatment of the granulosa-lutein cells with 8-Br-cAMP, which stimulates cholesterol metabolism into progesterone. In contrast, steroidogenic acute regulatory (StAR) protein levels were increased by 8-Br-cAMP. Incubation of granulosa-lutein cells with low-density lipoprotein (LDL) in the presence of the hydrophobic amine, U18666A, caused accumulation of free cholesterol in granules, identified by filipin staining, that contained LAMP-2 and NPC1. These granules also stained for neutral lipid with Nile red, reflecting accumulation of LDL-derived cholesterol esters. LDL-stimulated progesterone synthesis was completely blocked by U18666A, leaving steroid output at levels similar to those of cells incubated in the absence of LDL. The hydrophobic amine also blocked the LDL augmentation of 8-Br-cAMP-stimulated progesterone synthesis, reducing steroid production to levels seen in cells stimulated with 8-Br-cAMP in the absence of LDL. Steroidogenesis recovered after U18666A was removed from the culture medium. U18666A treatment caused a 2-fold or more increase in NPC1 protein and mRNA levels, suggesting that disruption of NPC1's function activates a compensatory mechanism resulting in increased NPC1 synthesis. We conclude that the NPC1 compartment plays an important role in the trafficking of LDL-derived substrate in steroidogenic cells; that NPC1 expression is up-regulated when NPC1 action is blocked; and that the NPC1 compartment can be functionally separated from other intracellular pathways contributing substrate for steroidogenesis.     

XBP1S Associates with RUNX2 and Regulates Chondrocyte Hypertrophy

BMP2 (bone morphogenetic protein 2) is known to activate unfolded protein response signaling molecules, including XBP1S and ATF6. However, the influence on XBP1S and ATF6 in BMP2-induced chondrocyte differentiation has not yet been elucidated. In this study, we demonstrate that BMP2 mediates mild endoplasmic reticulum stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. XBP1S is differentially expressed during BMP2-stimulated chondrocyte differentiation and exhibits prominent expression in growth plate chondrocytes. This expression is probably due to the activation of the XBP1 gene by ATF6 and splicing by IRE1a. ATF6 directly binds to the 5′-flanking regulatory region of the XBP1 gene at its consensus binding elements. Overexpression of XBP1S accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type II collagen, type X collagen, and RUNX2; however, knockdown of XBP1S via the RNAi approach abolishes hypertrophic chondrocyte differentiation. In addition, XBP1S associates with RUNX2 and enhances RUNX2-induced chondrocyte hypertrophy. Altered expression of XBP1S in chondrocyte hypertrophy was accompanied by altered levels of IHH (Indian hedgehog) and PTHrP (parathyroid hormone-related peptide). Collectively, XBP1S may be a novel regulator of hypertrophic chondrocyte differentiation by 1) acting as a cofactor of RUNX2 and 2) affecting IHH/PTHrP signaling.     

Early-phase redistribution of the cation-independent mannose 6-phosphate receptor by U18666A treatment in HeLa cells

It has been shown that the treatment with 3-[2-(diethylamino)ethoxy] androst-5-en-17-one (U18666A) causes the accumulation of cholesterol and the cation-independent mannose 6-phosphate receptor (CIMPR) in late endosomal/lysosomal compartments in BHK cells. The present study reports on a study of the effect of U18666A on CIMPR distribution in more detail in HeLa cells. When cells were treated with U18666A for 20 h, the intense perinuclear signal for CIMPR corresponding to the trans-Golgi network (TGN) disappeared and lamp1-negative punctate signals, scattered in the perinuclear region were detected. CIMPR then began to accumulate in lamp1-positive compartments 48 h after addition of the drug. Double immunofluorescence microscopy showed that U18666A-induced mannose 6-phosphate receptor-containing compartments (U-MPRCs), which were formed in the early phase of the redistribution, contained no marker for the TGN, late endosomes or lysosomes. Approximately half of the structures contained transferrin that had been internalized for 20 min, and cathepsin D, the majority of which appeared to be its precursor form. Immunoelectron-microscopic analysis revealed that U-MPRCs are composed of multivesicular bodies, irregularly shaped structures, and vesicular structures adjacent to the multivesicular bodies. These results suggest that U18666A treatment primarily suppresses the CIMPR transport pathways to late endosomes and from transferrin-containing endosomes, both of which may be dependent on cholesterol function.This work was supported by grants from Japan Ministry of Education, Culture, Sports, Science, and Technology.     

Identification of N-arachidonylglycine, U18666A, and 4-androstene-3,17-dione as novel insulin Secretagogues

The glucose-induced insulin secretion is fine-tuned by numerous factors. To systematically identify insulinotropic factors, we optimized a primary beta-cell-based functional assay to monitor intracellular Ca2+ flux ([Ca2+]i). By this assay system, we successfully identified several insulinotropic peptides including cholecystokinin, gastrin releasing peptide, vasopressin, and oxytocin from tissue extracts. Screening of an assortment of chemical compounds, we determined three novel insulin secretagogues: N-arachidonylglycine (NAGly), 3beta-(2-diethylamino-ethoxy) androstenone hydrochloride (U18666A), and 4-androstene-3,17-dione. The NAGly increased [Ca2+]i through stimulation of the voltage-dependent Ca2+ channels and it was dependent on extracellular glucose level. On the other hand, U18666A and 4-androstene-3,17-dione increased [Ca2+]i in the presence of K ATP channel opener diazoxide while it was inhibited by the presence of Ca2+ channel blocker nitrendipine, suggesting that their effects are independent of K ATP channel. These unique features will be useful for further development of insulinotropic factors and drugs for treating type 2 diabetes.     

The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity

We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.     

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin-stimulated proliferation of vascular smooth muscle cells

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA₂) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA₂ are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA₂ and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA₂ by 1.5-fold (p < 0.01), which was blocked by the cPLA₂ inhibitor MAFP (10 μM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 μM; 15 min) and ERK1/2 inhibitor PD98059 (20 μM; 15 min) abolished the INS-mediated increase in cPLA₂ phosphorylation by 59% (p < 0.001), and by 75% (p < 0.001), respectively. Further, inhibition of cPLA₂ with cPLA₂ inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% (p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% (p < 0.001). The effect of INS on VSMCs proliferation was significantly (p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA₂ and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA₂ activity.     

In vivo effects of human recombinant tumor necrosis factor alone and in combination with other biological response modifiers on human digestive organ cancer xenografts transplanted in nude mice

The present study was designed to evaluate the effect of rTNF alone or in combination with other BRMs on human digestive organ cancers. Six kinds of human digestive organ cancer xenografts (esophageal, stomach, colonic, pancreatic, bile duct, and liver cancers: EC-YO, GC-YN, CC-KK, PC-HN, BDC-SN and Li-7, respectively) were transplanted in nude mice, and rTNF was administered at 10³, 5 × 10³, or 10⁴U/head directly into the tumor 3 times a week for 2 weeks. EC-YO was the most sensitive to rTNF, and intratumoral administration of rTNF at 10³ U/head caused tumor regression. PC-HN, CC-KK and GC-YN were relatively sensitive to rTNF, and their growth was significantly inhibited by rTNF at 5 × 10³ U/head, however, the tumors regrew after treatment. Li-7 and BDC-SN were resistant to rTNF. The effects of rTNF in combination with recombinant interferon- (rIFN-), recombinant interleukin-2 (rIL-2), or streptococcal preparation OK-432 were assessed in mice transplanted with GC-YN. All combinations of rTNF at 5 × 10³ U/head and other BRMs were more effective than rTNF alone, and GC-YN tumors were completely regressed after treatment with a combination of rTNF and rIFN- or rTNF and OK-432. However in all cases, the combination of rTNF at 10³ U/head and any other BRM did not improve the effect. Furthermore, the adverse effects of the combinations were more serious than those of rTNF alone.TNF may still be a useful cytokine, because it can induce the regression of tumors. However, for its clinical application, a method should be developed to reduce its side effects.