Influence of oxidized low-density lipoproteins (LDL) on the viability of osteoblastic cells

Cardiovascular diseases have recently been noted as potential risk factors for osteoporosis development. Although it is poorly understood how these two pathologies are related, it is a known fact that oxidized low-density lipoproteins (OxLDL) constitute potential determinants for both of them. The current study investigated the metabolism of OxLDL by osteoblasts and its effect on osteoblastic viability. The results obtained show that OxLDL are internalized but not degraded by osteoblasts while they can selectively transfer their CE to these cells. It is also demonstrated that OxLDL induce proliferation at low concentrations but cell death at high concentrations. This reduction of osteoblast viability was associated with lysosomal membrane damage caused by OxLDL as demonstrated by acridine orange relocalization. Accordingly, chloroquine, an inhibitor of lysosomal activity, accentuated cell death induced by OxLDL. Finally, we demonstrate that osteoblasts have the capacity to oxidize LDL and thereby potentially increase the local concentration of OxLDL. Overall, the current study confirms the potential role of OxLDL in the development of osteoporosis given its influence on osteoblastic viability.     

Phorbol esters alter muscarinic receptor binding and inhibit polyphosphoinositide breakdown in human neuroblastoma (SH-SY5Y) cells

Many recent reports have indicated that the effect of the phorbol ester tumor promoters is mediated through the Ca2+/phospholipid dependent protein kinase C. We have investigated the effect of two biologically active phorbol esters, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) and 4 beta-phorbol 12 beta,13 alpha-didecanoate (beta PDD) on muscarinic agonist binding and receptor-stimulated phosphoinositide breakdown in cultured human neuroblastoma (SH-SY5Y) cells. Preincubation of these cells with phorbol esters significantly reduced the carbachol-stimulated breakdown of inositol phospholipids and caused a decrease of agonist affinity for [3H](-)methyl quinuclidinyl benzilate ([3H](-)MQNB) binding without affecting the affinity of antagonist to the muscarinic receptor. The nontumor promoting 4 alpha-phorbol 12 beta,12 alpha-didecanoate (alpha PDD) was ineffective in our studies. These results suggest that the activation of protein kinase C may play an important role in regulating the muscarinic receptor system.     

Degradation of low-density lipoproteins (LDL) and LDL - protamine complexes by lysosomal protease.

Native LDL are degraded by the protease of the lysosomal extract but they are not sensitive to isolated cathepsin D. Protamine increases the sensitivity of LDL to the effect of lysosomal protease and makes them sensitive to the effect of cathepsin D. Degradation of LDL by lysosomal protease is most intensive between pH 4.0 and 4.5 but in case of LDL bound with protamine it is most intensive at pH 4.5--5.0.     

Phorbol esters inhibit the synthesis of acetylcholine receptors in cultured muscle cells

The acetylcholine receptor (AChR) synthesis, insertion and degradation rates are regulated by numerous intracellular and extracellular agents. Recent studies have shown that Ca2+ and Ca2+ ionophores have a profound regulatory effect on the appearance of AChR clusters and AChR synthesis. These regulatory effects may be mediated through the activation of calcium and phospholipid-dependent protein kinases by agents such as phorbol esters. In this study, we have utilized 4-beta-phorbol-12-myristate-13-acetate (PMA) in order to determine whether the activation of protein kinase C exerts a regulatory effect on the expression of AChRs in cultured chick myotubes. Our results show that 4-beta-phorbol-12-myristate-13-acetate decreased intracellular AChRs and suppressed AChR synthesis without affecting the turnover rate. Control and PMA treated cells labeled with [35S] methionine and immunoprecipitated with a monoclonal antibody to the alpha subunit of AChRs (mAb35) revealed a significant decrease in radioactivity precipitated after exposure to PMA. Polyacrylamide gel electrophoresis revealed no major changes in protein patterns, or in newly synthesized proteins as determined by [35S] methionine incorporation and autoradiography. Other enzymes important in muscle metabolism were not affected by PMA treatment. Our results indicate that activation of protein kinase C results in the suppression of AChRs synthesis and dispersal of AChR clusters.     

Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36

Transmembrane protein CD36 binds multiple ligands, including oxidized low-density lipoproteins (oxLDLs) and long-chain fatty acids (LCFAs). Our aim was to determine whether LCFAs compete with oxLDLs for binding to CD36. We addressed this issue by examining the inhibitory effect of LCFAs against the binding of Alexa-fluor-labeled oxLDLs (AFL-oxLDL) to a synthetic peptide representing the oxLDL-binding site on CD36 (3S-CD36_150–168). All of the unsaturated LCFAs tested, inhibited the binding of AFL-oxLDL to 3S-CD36_150–168, albeit to varying degrees. For instance, the concentrations required for 50% inhibition of binding for oleic, linoleic, and α-linolenic acids were 0.25, 0.97, and 1.2?mM, respectively. None of the saturated LCFAs tested (e.g. stearic acid) exhibited inhibitory effects. These results suggest that at least unsaturated LCFAs can compete with oxLDLs for binding to CD36. The study also provides information on the structural requirements of LCFAs for inhibition of oxLDLs–CD36 binding.     

Apolipoprotein C-I reduces cholesteryl esters selective uptake from LDL and HDL by binding to HepG2 cells and lipoproteins

Plasma cholesterol from low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors that either totally degrade lipoproteins as the LDL receptor or selectively take up their cholesteryl esters (CE) like the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-I on the uptake of LDL and HDL₃ by HepG2 cells. In experiments conducted with exogenously added purified apoC-I, no significant effect was observed on lipoprotein–protein association and degradation; however, LDL- and HDL₃-CE selective uptake was significantly reduced in a dose-dependent manner. This study also shows that apoC-I has the ability to associate with HepG2 cells and with LDL and HDL₃. Moreover, pre-incubation of HepG2 cells with apoC-I reduces HDL₃-CE selective uptake and pre-incubation of LDL and HDL₃ with apoC-I decreases their CE selective uptake by HepG2 cells. Thus, apoC-I can accomplish its inhibitory effect on SR-BI activity by either binding to SR-BI or lipoproteins. We conclude that by reducing hepatic lipoprotein-CE selective uptake, apoC-I has an atherogenic character.     

Phorbol esters inhibit LH stimulated steroidogenesis by mouse Leydig cells in vitro

The effect of phorbol esters on the stimulation of testosterone production in response to LH was studied in mouse Leydig cells incubated in vitro. The tumor promoting phorbol esters, Phorbol-12-myristate-13-acetate and Phorbol-12-13-didecanoate at nanomolar concentrations effectively inhibited testosterone production by Leydig cells in response to stimulation by LH, whereas non-tumor promoting phorbol esters were ineffective. When the cells were stimulated by 8Br-cAMP, instead of LH, the testosterone production was stimulated similarly as in the presence of LH, but phorbol esters were without any effect. This suggests that the tumor promoting phorbol esters may act in the Leydig cells by suppressing the stimulation of cAMP production in response to hormonal activation and/or by interfering with the hormone-receptor interaction.     

Morphological studies on the binding of low-density lipoproteins and acetylated low-density lipoproteins to the plasma membrane of cultured monocytes

Binding of low density lipoproteins (LDL) and acetyl-LDL to the plasma membrane of cultured swine monocytes was investigated by immunofluorescent and immunoelectron microscopy. Binding sites for native LDL, visualized on both the light microscopical and the ultrastructural level, were found to be comparable to those of cultured human fibroblasts. These techniques, however, failed to reveal binding of acetyl-LDL to the cell surface. Biochemical experiments showed that both LDL and acetyl-LDL have specific receptors, the acetyl-LDL receptor being distinctly different from the LDL receptor. It is concluded that there are morphological differences in the binding of LDL and acetyl-LDL to cultured monocytes. These differences are supported by biochemical data.     

Stimulation of mitogen activated protein kinase by LDL and oxLDL in human U-937 macrophage-like cells

Mitogen activated protein kinase in extracts of U-937 macrophage-like cells was stimulated by LDL and oxLDL. A maximum value (161% of the basal phosphotransferase activity) was obtained after 6 min exposure to oxidized LDL (27 μ/ml) using APRTPGGRR peptide substrate. The activatory effect was more pronounced (LDL 181%, oxLDL 201%) when MAPK of stimulated cells was immunoprecipitated with anti-p42^MAPK antibodies and phosphotransferase activity was assayed in immune complexes. Stimulation produced by oxLDL was inhibited by poly I, fucoidan, dextran sulfate and by the MAPKK inhibitor PD 098059 but not by PMA-mediated depletion of PKC or by pre-treatment with chloroquine or with pertussis toxin. These results suggest a direct mitogenic effect of LDL which, in the case of oxLDL, is dependent on scavenger receptor ligation but not on G-protein mediated or PKC-dependent signal transduction.     

Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.     

Phorbol esters, but not the hormonal form of vitamin D, induce changes in protein kinase C during differentiation of human histiocytic lymphoma cell line (U-937)

Human histiocytic lymphoma cells (U-937) undergo similar differentiation when incubated with the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) and 1,25-dihydroxycholecalciferol. In this action, TPA somehow implicates calcium-sensitive and phospholipid-dependent protein kinase (protein kinase C), which is rapidly and significantly affected by this inducer. On the contrary, 1,25-dihydroxycholecalciferol in its differentiating action does not involve protein kinase C thus suggesting that the secosteroid induces monocytic differentiation possible through a different mechanism of that of phorbol ester.     

Binding of low density lipoproteins (LDL) to platelets

Binding of LDL to platelets was studied by two independent methods, radioactive and flow cytofluorimetry, using 125I- and fluorescently labelled RITC-LDL. Saturation of 125I- and RITC-LDL binding to platelets, inhibition of binding by unlabelled LDL and a lower inhibitory effect of unlabelled HDL evidence the existence of a limited number of binding sites specific for LDL in platelets. Unlike nuclear cells platelets do not degrade LDL. The binding of LDL to platelets is reversible and independent of Ca2+. The decrease of total binding level at 4 degrees and the absence of heparin effect on the release of bound LDL suggest LDL incorporation into platelets.     

Phorbol esters inhibit inositol phosphate and diacylglycerol formation in proliferating HL60 cells. Relationship to differentiation

Phorbol 12-myristate 13-acetate (PMA) induces the differentiation of the human promyelocytic cell line, HL60, towards adherent macrophage-like cells within 2 days. We have examined the early effects of PMA on inositol phosphates and on diacylglycerol production, two second messengers derived from inositol lipids. In proliferating HL60 cells, PMA induced a time- and concentration-dependent decrease in inositol phosphate levels. Maximal effects were seen after 1 h at 10 nM PMA. PMA also induced the translocation of protein kinase C from the cytosol to the membrane. Comparison between the differentiating effects of several phorbol esters and of 1-oleoyl-2-acetylglycerol with their ability to inhibit inositol phosphate formation suggests that the two effects are correlated.     

Phorbol esters inhibit phosphate uptake in opossum kidney cells: a model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.