Osteoprotegerin ligand modulates murine osteoclast survival in vitro and in vivo.

Osteoprotegerin ligand (OPGL) targets osteoclast precursors and osteoclasts to enhance differentiation and activation, however, little is known about OPGL effects on osteoclast survival. In vitro, the combination of OPGL + colony-stimulating factor-1 (CSF-1) is required for optimal osteoclast survival. Ultrastructurally, apoptotic changes were observed in detached cells and culture lysates exhibited elevated caspase 3 activity, particularly in cultures lacking CSF-1. DEVD-FMK (caspase 3 inhibitor) partially protected cells when combined with OPGL, but not when used alone or in combination with CSF-1. CSF-1 maintained NF-kappaB activation and increased the expression of bcl-2 and bcl-X(L) mRNA, but had no effect on JNK activation. In contrast, OPGL enhanced both NF-kappaB and JNK kinase activation and increased the expression of c-src, but not bcl-2 and bcl-X(L) mRNA. These data suggest that aspects of both OPGL's and CSF-1's signaling/survival pathways are required for optimal osteoclast survival. In mice, a single dose of OPG, the OPGL decoy receptor, led to a >90% loss of osteoclasts because of apoptosis within 48 hours of exposure without impacting osteoclast precursor cells. Therefore, OPGL is essential, but not sufficient, for osteoclast survival and endogenous CSF-1 levels are insufficient to maintain osteoclast viability in the absence of OPGL.

On the complexities of ceramide changes in cells undergoing apoptosis: lack of evidence for a second messenger function in apoptotic induction.

The generation of cellular ceramides as a second messenger has been implicated as a regulatory and required step for the induction of apoptosis. In this study, we have applied a recently developed mass spectrometric technique to the determination of changes in physiological ceramide levels during apoptosis induced by tumor necrosis factor plus cycloheximide in U937 cells and the chemical agents anisomycin or geranylgeraniol in HL-60 cells. The mass spectrometric method has significant advantages over traditional methods for ceramide quantitation in that it determines the relative abundance of all ceramide species present in complex biological lipid mixtures individually and simultaneously. We quantitiated ceramides ranging from C14 to C26, finding that their basal levels and relative distribution varied significantly, both within and between different cell types. However, we were not able to detect any significant changes in either total ceramide content or species distribution until 1 h or more post-stimulation with any of these treatments, by which time the cells were in an advanced stage of apoptosis. Differences were also seen between all three treatments in the ceramide species distribution observed in these late stages of apoptosis. These data indicate that in vivo ceramide generation occurs as a consequence of apoptosis rather than as an essential second messenger involved in its induction. They also pose new questions about the potential roles that certain ceramide species may play in the late stages of apoptosis, and demonstrate a clear need to utilize the resolving power of mass spectrometry-based assays in any future investigations into the biological function of ceramides.

Fas-induced apoptosis of T cells occurs independently of ceramide generation.

The Fas receptor is one of a number of important physiological inducers of programmed cell death (apoptosis). Current models for regulation of this process involve rapid conversion of sphingomyelin to ceramide by cellular sphingomyelinases. Induced changes in cellular levels of such sphingosine-based ceramides are normally extrapolated from measurements of sphingomyelinase activity or following their conversion to ceramide phosphate by treatment of cellular lipid extracts with bacterial diacylglycerol kinase (DAGK). To allow direct study of cellular sphingosine- and sphinganine-based ceramide levels, we developed a mass spectrometric technique capable of determining inducible changes in both overall ceramide levels and species distribution in cellular lipid preparations. Contrary to current models, we detected no changes in cellular ceramide levels up to 2 hr poststimulation of Jurkat T cells with an anti-Fas IgM, although this treatment did induce apoptosis. We also determined in the same system that, when utilizing the DAGK assay, increased phosphorylation of substrates that comigrated with ceramide standards was apparent but that this effect was due to an enhancement of DAGK activity rather than increases in levels of cellular ceramides as substrates per se. Thus, the first direct measurement of ceramides present in cells undergoing apoptosis indicates that, insofar as it can be measured, the induction of apoptosis does not involve the generation of sphingosine-based ceramides, contrary to many published accounts.

Selective activation of caspases during apoptotic induction in HL-60 cells. Effects Of a tetrapeptide inhibitor.

Apoptosis is a highly regulated biochemical process that results in the selective death of cells. Members of the caspase family of cysteine proteases play a pivotal role in the effector phase of apoptosis. We show that, in HL-60 cells, the addition of either anisomycin, a protein synthesis inhibitor, or geranylgeraniol, an intermediate in the cholesterol biosynthetic pathway, results in a rapid and en masse induction of apoptosis. The levels of actin, p42 and p44 MAPK, JNK1, JNK2, p38, and PCNA were not substantially altered during this process. Although these treatments appear to function by diverse pathways, they both result in the processing and activation of caspase-3 (CPP32beta/Yama/Apopain). In contrast, no activation of caspase-1 (interleukin-1beta converting enzyme (ICE)) was observed. Furthermore, we obtained ambiguous results regarding the activation of caspase-2 (Ich-1) depending on the antibody used. Pretreatment of the cells with benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD.fmk), a tetrapeptide inhibitor of caspases, prevented the induction of apoptosis for 24 h. Even after 72 h of treatment, some cells were still alive and progressing through the cell cycle, suggesting that blockage of caspase activity is able to protect cells. These results suggest that selective activation of some caspases is necessary to induce apoptosis in HL-60 cells.

Oncogenic ras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescent Xenopus oocytes.

Quiescent, full-grown Xenopus oocytes, which are arrested at the G2/M border of meiosis, contain an inactive 42-kDa mitogen-activated protein kinase (p42MAPK) that is activated when oocytes are stimulated to resume the meiotic cell cycle. We have made extracts from these oocytes that respond to four cell cycle activators: oncogenic [Val12]Ras protein, clam cyclins A delta 60 and B delta 97, and the phosphatase inhibitor okadaic acid. All four induce the tyrosine phosphorylation and activation of p42MAPK. Both cyclins and okadaic acid, but not [Val12]Ras, also lead to activation of the endogenous cyclin B/cdc2 kinase complexes in extracts of quiescent oocytes. Using extracts prepared from cycloheximide-arrested interphase cells, we show that although p42MAPK activation can occur in response to cyclin-activated cdc2, the Ras-induced activation of p42MAPK occurs without intervening cdc2 activation. Neither the nononcogenic [Gly12]Ras nor [Val12,Arg186]Ras, a mutant that lacks the C-terminal consensus sequence directing prenylation and subsequent membrane association, is an effective activator of p42MAPK in vitro.

Inhibition of Ca2+ inflow causes an abrupt cessation of growth-factor-induced repetitive free Ca2+ transients in single NIH-3T3 cells.

In single NIH-3T3 fibroblasts loaded with fura-2, bombesin induced one of three patterns of increase in the concentration of intracellular free Ca2+ [( Ca2+]i): a single transient increase, a sustained increase, or repetitive transient increases in [Ca2+]i. Foetal-calf serum and ATP also gave these three patterns of response, although a lower proportion of cells gave repetitive Ca2+ transients in response to ATP. An increase in the concentration of bombesin from 1 to 25 nM increased the proportion of cells which exhibited repetitive Ca2+ transients. At 25 nM-bombesin, the proportion of cells which exhibited repetitive Ca2+ transients increased as the extracellular Ca2+ (Ca2+o) concentration was increased from 1 to 5 mM. Removal of Ca2+o by addition of EGTA, or inhibition of Ca2+ inflow by treatment of cells incubated in the presence of Ca2+o with verapamil or an activator of protein kinase C, abruptly terminated repetitive Ca2+ transients, with only one transient observed after the cessation of Ca2+ inflow. Repetitive Ca2+ transients were not observed in cells incubated in the absence of Ca2+o and in the presence of EGTA. Addition of Ca2+o to cells previously incubated in the presence of EGTA caused a resumption of repetitive Ca2+ transients. Addition of thapsigargin alone induced a large transient increase in [Ca2+]i, whereas much smaller transient increases in [Ca2+]i were induced in about 30% of cells tested by caffeine or carbonyl cyanide m-chlorophenylhydrazone (CCCP) plus oligomycin. Thapsigargin or the combination of CCCP plus oligomycin completely inhibited bombesin-induced repetitive Ca2+ transients, whereas caffeine had no effect. It is concluded from the studies of the role of Ca2+o that NIH-3T3 cells differ from other cell types in the anatomical or chemical links between extracellular Ca2+ and the intracellular stores involved in the generation of Ca2+ transients, whereas the results of the experiments with inhibitors indicate that the generation of repetitive Ca2+ transients in NIH-3T3 cells is unlikely to involve Ca(2+)-induced Ca2+ release from caffeine-sensitive stores.

NIH-3T3 cells transformed with a ras oncogene exhibit a protein kinase C-mediated inhibition of agonist-stimulated Ca2+ inflow.

1. The ability of bombesin or platelet-derived growth factor (PDGF) to stimulate Ca2+ inflow (assessed by measuring changes in the intracellular free Ca2+ concentration in cells loaded with fura-2) in NIH-3T3 cells transformed with the EJ/T24-Ha-ras-1 oncogene is inhibited when compared with the action of the agonists on wild-type cells. 2. The effects of transformation with the ras oncogene are associated with complete inhibition of the ability of bombesin to release Ca2+ from intracellular stores, a substantial decrease in the number of bombesin receptors, no change in the ability of foetal calf serum or ionomycin to release Ca2+ from intracellular stores and the activation of protein kinase C. 3. The effects of transformation with the H-ras oncogene on the ability of bombesin or PDGF to stimulate Ca2+ inflow were mimicked by a 30 min exposure of wild-type cells to phorbol dibutyrate. This action of phorbol dibutyrate was completely blocked by prior treatment of wild-type cells for 24 h with the phorbol ester. 4. It is concluded that one of the actions of the H-ras oncogene in fibroblasts is to inhibit agonist-stimulated Ca2+ inflow by a mechanism which involves the activation of protein kinase C.

On the source of the vasopressin-induced increases in diacylglycerol in hepatocytes.

In hepatocytes pre-labelled with [3H]glycerol, vasopressin increased by 20% the amount of radioactivity present in diacylglycerols. The effect of vasopressin was partially dependent on Ca2+. The magnitude of the increase in [3H]diacylglycerol was 5-times the sum of the radioactivity present in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. No stimulation by vasopressin of the initial rate of incorporation of radioactivity into diacylglycerols was observed in cells incubated in the presence of 10 mM [3H]glycerol. Treatment of hepatocytes labelled with either [3H]ethanolamine or [3H]choline with vasopressin, ionophore A23187 or phospholipase C increased the amount of radioactivity present in trichloroacetic acid extracts of the cells. The effect of vasopressin was dependent on extracellular Ca2+. It is concluded that in hepatocytes vasopressin increases diacylglycerols by a process which does not principally involve the conversion of phosphoinositides to diacylglycerol or the de novo synthesis of diacylglycerol from glycerol 3-phosphate, but does involve the Ca2+-dependent conversion of phosphatidylethanolamine and phosphatidylcholine to diacylglycerol.

Evidence from studies employing radioactively labelled fatty acids that the stimulation of flux through the diacylglycerol pool is an early action of vasopressin on hepatocytes.

1. In isolated hepatocytes prelabelled with [14C]-arachidonic, -stearic, -linoleic, -oleic or -palmitic acids, vasopressin increased the amount of radioactivity present in diacylglycerols. The largest increase was observed in cells labelled with arachidonic or stearic acids. 2. In cells prelabelled with [14C]- or [3H]-arachidonic acid, the onset of the increase in radioactivity in diacylglycerols induced by vasopressin was slow, the increase was partly dependent on the presence of extracellular Ca2+, and was associated with an increase in radioactivity present in phosphatidic acid which was more rapid in onset. Vasopressin decreased the amount of [3H]arachidonyl-phosphatidylinositol 4,5-bisphosphate, but the magnitude of this decrease was less than 10% of the observed increase in radioactivity in [3H]arachidonyl-diacylglycerol. 3. The concentration of vasopressin which gave half-maximal increase in [14C]arachidonyl-diacylglycerol at low extracellular Ca2+ was 10-fold higher than that which gave half-maximal stimulation of 45Ca2+ efflux. Phenylephrine, but not glucagon, also increased the amount of [14C]arachidonyl-diacylglycerol. 4. It is concluded that an early action of vasopressin on the liver cell is to increase the flux of carbon from phospholipids, including the phosphoinositides, to diacylglycerols.

Vasopressin decreases total free fatty acids but enhances release of radioactivity from isolated hepatocytes labelled with [3H]arachidonic acid.

The short-term effects of vasopressin on free fatty acids and lysophospholipids were investigated in hepatocytes isolated from fed rats. Over the time period 0.25 to 10 min vasopressin decreased the steady-state concentrations of palmitic, stearic and oleic acids measured by gas liquid chromatography in extracts of cells incubated at 0.1 mM extracellular Ca2+. The concentrations of arachidonic and linoleic acids did not change. In hepatocytes labelled with [3H]arachidonic acid and incubated at 1.3 mM extracellular Ca2+ vasopressin or the Ca2+-selective ionophore A23187 increased the rate of accumulation of radioactivity in the incubation medium by 40%. The action of A23187 was dependent on extracellular Ca2+. When hepatocytes labelled with 32Pi were treated with vasopressin, no change in the amounts of [32P]lysophosphatidylethanolamine or [32P]lysophosphatidylcholine was observed. It is concluded that the action of vasopressin on hepatocytes is associated with the release of arachidonic acid or metabolites of arachidonic acid but is not accompanied by a general increase in the steady-state concentrations of free fatty acids and lysophospholipids.