Sperm factor induces intracellular free calcium oscillations by stimulating the phosphoinositide pathway

Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca(2+)](i)) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca(2+)](i) oscillations, although it did not inhibit Ca(2+) release caused by injection of inositol 1,4,5-triphosphate (IP(3)). The inactive analogue, U73343, had no effect on SF-induced Ca(2+) responses. To determine at the single cell level whether SF triggers IP(3) production concomitantly with a [Ca(2+)](i) rise, SF was injected into Xenopus oocytes and IP(3) concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca(2+)](i) and IP(3) production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca(2+) activity in the extracts. Our results revealed that active fractions do not contain PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP(3) could be the sensitizing stimulus of the Ca(2+)-induced Ca(2+) release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP(3) receptor agonist, showed enhanced Ca(2+) responses to CaCl(2) injections. Thus, SF, and probably sperm, induces [Ca(2+)](i) rises by persistently stimulating IP(3) production, which in turn results in long-lasting sensitization of Ca(2+)-induced Ca(2+) release. Whether SF is itself a PLC or whether it acts upstream of the egg's PLCs remains to be elucidated.     

Lipocalin-2 induces cardiomyocyte apoptosis by increasing intracellular iron accumulation

Our objective was to determine whether lipocalin-2 (Lcn2) regulates cardiomyocyte apoptosis, the mechanisms involved, and the functional significance. Emerging evidence suggests that Lcn2 is a proinflammatory adipokine associated with insulin resistance and obesity-related complications, such as heart failure. Here, we used both primary neonatal rat cardiomyocytes and H9c2 cells and demonstrated for the first time that Lcn2 directly induced cardiomyocyte apoptosis, an important component of cardiac remodeling leading to heart failure. This was shown by detection of DNA fragmentation using TUNEL assay, phosphatidylserine exposure using flow cytometry to detect annexin V-positive cells, caspase-3 activity using enzymatic assay and immunofluorescence, and Western blotting for the detection of cleaved caspase-3. We also observed that Lcn2 caused translocation of the proapoptotic protein Bax to mitochondria and disruption of mitochondrial membrane potential. Using transient transfection of GFP-Bax, we confirmed that Lcn2 induced co-localization of Bax with MitoTracker® dye. Importantly, we used the fluorescent probe Phen Green SK to demonstrate an increase in intracellular iron in response to Lcn2, and depleting intracellular iron using an iron chelator prevented Lcn2-induced cardiomyocyte apoptosis. Administration of recombinant Lcn2 to mice for 14 days increased cardiomyocyte apoptosis as well as an acute inflammatory response with compensatory changes in cardiac functional parameters. In conclusion, Lcn2-induced cardiomyocyte apoptosis is of physiological significance and occurs via a mechanism involving elevated intracellular iron levels and Bax translocation.     

Static pressure accelerates ox-LDL-induced cholesterol accumulation via SREBP-1-mediated caveolin-1 downregulation in cultured vascular smooth muscle cells

Objective

To investigate the effect of static pressure on cholesterol accumulation in vascular smooth muscle cells (VSMCs) and its mechanism.

Methods

Rat-derived VSMC cell line A10 treated with 50 mg/L ox-LDL and different static pressures (0, 60, 90, 120, 150, 180 mm Hg) in a custom-made pressure incubator for 48 h. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC; The mRNA levels of caveolin-1 and ABCA1, the protein levels of caveolin-1 SREBP-1 and mature SREBP-1 were respectively detected by RT-PCR or western blot. ALLN, an inhibitor of SREBP metabolism, was used to elevate SREBP-1 protein level in VSMCs treated with static pressure.

Results

Static pressures significantly not only increase intracellular lipid droplets in VSMCs, but also elevate cellular lipid content in a pressure-dependent manner. Intracellular free cholesterol (FC), cholesterol ester (CE), total cholesterol (TC) were respectively increased from 60.5 ± 2.8 mg/g, 31.8 ± 0.7 mg/g, 92.3 ± 2.1 mg/g at atmosphere pressure (ATM, 0 mm Hg) to 150.8 ± 9.4 mg/g, 235.9 ± 3.0 mg/g, 386.7 ± 6.4 mg/g at 180 mm Hg. At the same time, static pressures decrease the mRNA and protein levels of caveolin-1, and induce the activation and nuclear translocation of SREBP-1. ALLN increases the protein level of mature SREBP-1 and decreases caveolin-1 expression, so that cellular lipid levels were upregulated.

Conclusion

Static pressures stimulate ox-LDL-induced cholesterol accumulation in cultured VSMCs through decreasing caveolin-1 expression via inducing the maturation and nuclear translocation of SREBP-1.     

Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells.

Oxidation of low density lipoprotein (LDL) by cells of the arterial wall or in the presence of copper ions was shown to result in the peroxidation of its fatty acids as well as its cholesterol moiety. LDL incubation with cholesterol oxidase (CO) resulted in the conversion of up to 85% of the lipoprotein unesterified cholesterol (cholest-5-en-3-ol) to cholestenone (cholest-4-en-3-one) in a dose- and time-dependent pattern. Plasma very low density lipoprotein (VLDL) and high density lipoprotein (HDL) could be similarly modified by CO. In cholesterol oxidase-modified LDL (CO-LDL), unlike copper ion-induced oxidized LDL (Cu-Ox-LDL), there was no fatty acids peroxidation, and lipoprotein size or charge as well as LDL cholesteryl ester, phospholipids, and triglycerides content were not affected. CO-LDL, however, demonstrated enhanced susceptibility to oxidation by copper ions in comparison to native LDL. Upon incubation of CO-LDL with J-774 A.1 macrophage-like cell line, cellular uptake and degradation of the lipoprotein was increased by up to 62% in comparison to native LDL but was 15% lower than that of Cu-Ox-LDL. Similarly, the binding of CO-LDL to macrophages increased by up to 80%, and cellular cholesterol mass was increased 51% more than the mass obtained with native LDL. Several lines of evidence indicate that CO-LDL was taken up via the LDL receptor: 1) Excess amounts of unlabeled LDL, but not acetyl-LDL (Ac-LDL), effectively competed with 125I-CO-LDL for the uptake by cells. 2) The degradation of CO-LDL by various types of macrophages and by fibroblasts could be dissociated from that of Ac-LDL and was always higher than that of native LDL. 3) A monoclonal antibody to the LDL receptor (IgG-C7) and a monoclonal antibody to the LDL receptor binding domains on apoB-100 (B1B6) inhibited macrophage degradation of CO-LDL. The receptor for Cu-Ox-LDL, which is not shared with Ac-LDL, was also partially involved in macrophage uptake of CO-LDL, since Cu-Ox-LDL demonstrated some competition capability with CO-125I-LDL for its cellular degradation. CO-LDL cellular degradation was inhibited by chloroquine, thus implying lysosomal involvement in the cellular processing of the lipoprotein. Incubation of macrophages with LDL in the presence of increasing concentrations of cholestenone resulted in up to 52% enhanced lipoprotein cellular degradation suggesting that the cholestenone in CO-LDL might be involved in the enhanced cellular uptake of the modified lipoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

BMAL1 induces colorectal cancer metastasis by stimulating exosome secretion

Molecular Biology Reports - To adapt to daily changes in the external environment, organisms have developed circadian rhythm systems with a period of approximately 24 h. Many studies have...     

LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling

The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.     

Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1

Intracellular accumulation of mutant Huntingtin with expanded polyglutamine provides a context-dependent cytotoxicity critical for the pathogenesis of Huntington disease (Everett, C. M., and Wood, N. W. (2004) Brain 127, 2385-2405). Here we demonstrate that the accumulation of mutant Huntingtin is highly sensitive to the expression of beclin 1, a gene essential for autophagy. Moreover, we show that the accumulated mutant Huntingtin recruits Beclin 1 and impairs the Beclin 1-mediated long lived protein turnover. Thus, sequestration of Beclin 1 in the vulnerable neuronal population of Huntington disease patients might further reduce Beclin 1 function and autophagic degradation of mutant Huntingtin. Finally, we demonstrate that the expression of beclin 1 decreases in an age-dependent fashion in human brains. Because beclin 1 gene is haploid insufficient in regulating autophagosome function (Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., Cattoretti, G., and Levine, B. (2003) J. Clin. Invest. 112, 1809-1820; Yue, Z., Jin, S., Yang, C., Levine, A. J., and Heintz, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15077-15082), we propose that the age-dependent decrease of beclin 1 expression may lead to a reduction of autophagic activity during aging, which in turn promotes the accumulation of mutant Htt and the progression of the disease.     

SREBP-1 upregulates lipophagy to maintain cholesterol homeostasis in brain tumor cells

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Impaired recycling of apolipoprotein E4 is associated with intracellular cholesterol accumulation

After internalization of triglyceride-rich lipoproteins (TRL) in hepatoma cells, TRL particles are immediately disintegrated in the early endosomal compartment. This involves the targeting of lipids and apoprotein B along the degradative pathway and the recycling of TRL-derived apoE through recycling endosomes. Re-secretion of apoE is accompanied by the concomitant association of apoE and cellular cholesterol with high-density lipoproteins (HDL). Since epidemiological data showed that apoE3 and apoE4 have differential effects on HDL metabolism, we investigated whether the intracellular processing of TRL-derived apoE4 differs from apoE3-TRL. In this study, we demonstrated by radioactive and immunofluorescence uptake experiments that cell-surface binding and internalization of TRL-derived apoE4 are increased compared with apoE3 in hepatoma cells. Pulse-chase experiments revealed that HDL-induced recycling, but not disintegration and degradation, of apoE4-enriched TRL is strongly reduced in these cells. Furthermore, impaired HDL-induced apoE4 recycling is associated with reduced cholesterol efflux. Studies performed in Tangier fibroblasts showed that apoE recycling does not depend on ATP-binding cassette transporter A1 activity. These studies provide initial evidence that impaired recycling of apoE4 could interfere with intracellular cholesterol transport and contribute to the pathophysiological lipoprotein profile observed in apoE4 homozygotes.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Epigallocatechin gallate suppresses hepatic cholesterol synthesis by targeting SREBP-2 through SIRT1/FOXO1 signaling pathway

To investigate the mechanism of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) and human adipose-derived stem cells (hADSCs) in the treatment of Achilles tendinitis. Biomechanical indices of stiffness, stress, and maximum load-to-failure were detected by biomechanical test. mRNA and protein levels of miR-363, p-PI3K/AKT, tendon-related genes Collagen I, Scleraxis (Scx), and Tenascin C (TNC) were measured by qRT-PCR and western blot. The proliferation of hADSCs was accessed by MTT assay. Biomechanical indices of stiffness, stress, and maximum load-to-failure, and mRNA and protein levels of tendon-related genes could be improved by rhPDGF-BB or hADSCs alone, and could be further improved by rhPDGF-BB + hADSCs. rhPDGF-BB and hADSCs downregulated the expression of miR-363 and upregulated the levels of p-PI3K/Akt, and rhPDGF-BB + hADSCs further strengthened these effects. In addition, rhPDGF-BB promoted the proliferation of hADSCs in vitro and upregulated the expression of tendon-related genes. miR-363 mimic downregulated the levels of p-PI3K/Akt, miR-363 inhibitor upregulated the levels of p-PI3K/Akt, and miR-363 mimic and PI3K/Akt pathway inhibitor LY294002 reversed the positive effect of rhPDGF-BB on the proliferation of hADSCs, which suggested that rhPDGF-BB promoted the proliferation of hADSCs via miR-363/PI3K/Akt pathway. Biomechanical indices and tendon-related genes could be improved by rhPDGF-BB and hADSCs. Moreover, rhPDGF-BB promoted the proliferation of hADSCs via miR-363/PI3K/Akt pathway, indicating that rhPDGF-BB combined with ADSCs could treat Achilles tendinitis via miR-363/PI3K/Akt pathway.     

Modification of LDL by platelet secretory products induces enhanced uptake and cholesterol accumulation in macrophages

LDL modified by incubation with platelet secretory products caused cholesterol accumulation and stimulation of cholesterol esterification in mouse peritoneal macrophages. Its uptake by the macrophages was a receptor-mediated process, not susceptible to competition by acetyl-LDL or polyanions suggesting independence of the scavenger receptor. Stimulation of the esterification process in macrophages by this modified LDL was inhibited by the lysosomal inhibitor chloroquine, indicating requirement for cellular uptake and lysosomal hydrolysis of the lipoprotein. Within the cell, the modified LDL inhibited cellular biosynthesis of triglycerides in a manner similar to the action of acetyl-LDL but different to the effect of native LDL. In the presence of HDL, acting in the medium as an acceptor for cholesterol, a low rate of cholesterol efflux from cells incubated with this modified LDL as well as with acetyl-LDL was demonstrated. A small reduction in cholesteryl ester synthesis was found in these cells, compared to a 60% reduction in cells incubated with native LDL. Thus it was demonstrated that LDL modified by platelet secretory products could induce macrophage cholesterol accumulation even though it was recognized and taken up via the regulatory LDL receptor.     

Sphingolipid storage impairs autophagic clearance of Alzheimer-associated proteins

Recent work from our laboratory demonstrates that the accumulation of sphingolipids (SLs) decreases the capacity of cells to clear potentially amyloidogenic fragments of the amyloid precursor protein (APP) during autophagy. APP is a type I membrane protein and could undergo sequential proteolytic processing by β- and γ-secretase resulting in the generation of the amyloid β-peptide (Aβ). Genetic, molecular and biochemical evidence indicates that the accumulation of toxic Aβ aggregates plays a critical role in the degeneration of neurons during the pathogenesis of Alzheimer disease (AD). Thus, SL storage could promote the accumulation of Ab in endosomal and lysosomal compartments and thereby induce characteristic cytopathological changes of AD.     

Sphingolipid storage impairs autophagic clearance of Alzheimer-associated proteins

Recent work from our laboratory demonstrates that the accumulation of sphingolipids (SLs) decreases the capacity of cells to clear potentially amyloidogenic fragments of the amyloid precursor protein (APP) during autophagy. APP is a type I membrane protein and could undergo sequential proteolytic processing by β- and γ-secretase resulting in the generation of the amyloid β-peptide (Aβ). Genetic, molecular and biochemical evidence indicates that the accumulation of toxic Aβ aggregates plays a critical role in the degeneration of neurons during the pathogenesis of Alzheimer disease (AD). Thus, SL storage could promote the accumulation of Ab in endosomal and lysosomal compartments and thereby induce characteristic cytopathological changes of AD.