Stress-activated protein kinase/JNK activation and apoptotic induction by the macrophage P2X7 nucleotide receptor

In human and rodent macrophages, activation of the P2X7 nucleotide receptor stimulates interleukin-1beta processing and release, apoptosis, and killing of intracellular Mycobacterium tuberculosis. Signaling pathways downstream of this ionotropic ATP receptor are poorly understood. Here we describe the rapid activation of the stress-activated protein kinase (SAPK)/JNK pathway in BAC1 murine macrophages stimulated by extracellular ATP. Brief exposure of the cells to ATP (10-30 min) was sufficient to trigger a rapid accumulation of activated SAPK that was then sustained for >120 min. Several observations indicated that the P2X7 receptor mediated this effect. 1) ATP and 3'-O-(4-benzoyl)benzoyl-ATP were the only agonistic nucleotides. 2) The effect was inhibited by oxidized ATP and the isoquinoline KN-62, two known P2X7 receptor antagonists. 3) ATP-induced SAPK activation could be recapitulated in P2X7 receptor-transfected HEK293 cells, but not in wild-type HEK293 cells. Because P2X7 receptor stimulation can rapidly activate caspase family proteases that have been implicated in the induction of the SAPK pathway, we investigated whether ATP-dependent SAPK activation involved such proteases. Brief exposure of BAC1 macrophages to extracellular ATP induced DNA fragmentation, alpha-fodrin breakdown, and elevated levels of caspase-3-type activity. Asp-Glu-Val-Asp-cho, a caspase-3 inhibitor, inhibited ATP-induced DNA fragmentation and alpha-fodrin proteolysis, but had no effect on ATP-induced SAPK activation. Tyr-Val-Ala-Asp-chloromethyl ketone, a caspase-1 inhibitor, prevented ATP-induced release of processed interleukin-1beta, but not ATP-dependent SAPK activity. We conclude that activation of ionotropic P2X7 nucleotide receptors triggers a strong activation of SAPK via a pathway independent of caspase-1- or caspase-3-like proteases.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes

Plasma membrane wounds are repaired by a mechanism involving Ca(2+)-regulated exocytosis. Elevation in intracellular [Ca(2+)] triggers fusion of lysosomes with the plasma membrane, a process regulated by the lysosomal synaptotagmin isoform Syt VII. Here, we show that Ca(2+)-regulated exocytosis of lysosomes is required for the repair of plasma membrane disruptions. Lysosomal exocytosis and membrane resealing are inhibited by the recombinant Syt VII C(2)A domain or anti-Syt VII C(2)A antibodies, or by antibodies against the cytosolic domain of Lamp-1, which specifically aggregate lysosomes. We further demonstrate that lysosomal exocytosis mediates the resealing of primary skin fibroblasts wounded during the contraction of collagen matrices. These findings reveal a fundamental, novel role for lysosomes: as Ca(2+)-regulated exocytic compartments responsible for plasma membrane repair.     

Lysosomal involvement in hepatocyte cytotoxicity induced by Cu(2+) but not Cd(2+)

Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling.     

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.     

Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7

The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c-Myc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of c-Myc was also apparent in mouse Fbw7-/- embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively.     

Hydrogen peroxide-induced c-fos expression is mediated by arachidonic acid release: role of protein kinase C.

We found previously that stimulation of c-fos and c-myc mRNA expression are early events in hydrogen peroxide-induced growth in rat aortic smooth muscle (RASM) cells. In the present study, we investigated the role of phospholipase A2 (PLA2) and protein kinase C (PKC) in mediating hydrogen peroxide-induced c-fos mRNA expression in RASM cells. Mepacrine and p-bromophenacylbromide, potent inhibitors of PLA2 activity, blocked hydrogen peroxide-induced c-fos mRNA expression. Arachidonic acid, a product of PLA2 activity, stimulated the expression of c-fos mRNA with a time course similar to that of hydrogen peroxide. PKC down-regulation attenuated both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression by 50%. Nordihydroguaiaretic acid (a lipoxygenase-cytochrome P450 monooxygenase inhibitor) significantly inhibited both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Together, these findings indicate that 1) hydrogen peroxide-induced c-fos mRNA expression is mediated by PLA2-dependent arachidonic acid release, 2) both PKC-dependent and independent mechanisms are involved in hydrogen peroxide-induced expression of c-fos mRNA and 3) arachidonic acid metabolism via the lipoxygenase-cytochrome P450 monooxygenase pathway appears to be required for hydrogen peroxide-induced expression of c-fos mRNA.     

Nucleoplasmic Ca(2+)loading is regulated by mobilization of perinuclear Ca(2+)

Regulation of nucleoplasmic calcium (Ca(2+)) concentration may occur by the mobilization of perinuclear luminal Ca(2+)pools involving specific Ca(2+)pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca(2+), we examined freshly cultured 10 day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the existence of the molecular machinery required for the bi-directional Ca(2+)fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca(2+)channels (RyR2), sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca(2+)channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable to the nuclear envelope. Results obtained from Fluo-3, AM loaded ionomycin-perforated embryonic cardiomyocytes demonstrated that gradual increases of extranuclear Ca(2+)from 100 to 1600 nM Ca(2+)was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca(2+)loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca(2+)loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca(2+)loading at low concentrations of extranuclear added Ca(2+). Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca(2+)fluxes.     

Egr-1 is activated by 17beta-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1

Early growth response-1 (Egr-1) is an immediate-early gene induced by E2 in the rodent uterus and breast cancer cells. E2 induces Egr-1 mRNA and protein levels in MCF-7 human breast cancer cells and reporter gene activity in cells transfected with pEgr-1A, a construct containing the -600 to +12 region of the Egr-1 promoter linked to the firefly luciferase gene. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region of the promoter that contained serum response element (SRE)3 (-376 to -350) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a mitogen-activated protein kinase kinase inhibitor, but not by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3-K). These results contrasted with hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002. Differences in activation of the SREs in Egr-1 and c-fos were related to promoter sequence, which defines the affinities of Elk-1 and SRF to their respective binding sites. Thus, Egr-1, like c-fos, is activated through non-genomic (extranuclear) pathways of estrogen action in breast cancer cells.     

Short-term hibernation in adult cardiomyocytes is PO(2) dependent and Ca(2+) mediated

The mechanism of myocardial hibernation, the reversible downregulation of contractile activity on reduction of coronary flow with unchanged cardiac energetics, is presently not understood. The oxygen consumption (VO(2)), shortening fraction (DeltaL), energy status [phosphocreatine (PCr), ATP, and adenosine and lactate release], and free intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in isolated rat cardiomyocytes at precisely controlled ambient PO(2) (Oxystat). When PO(2) was reduced from 25 to 6 mmHg, VO(2) decreased by 50%, while DeltaL was downregulated from 11.2 +/- 4.1 to 7.6 +/- 4.0%, and energy status was unchanged in the steady state (observation time 12 min). Only transiently PCr decreased, and lactate and adenosine release increased. Further reduction of PO(2) (to 3 mmHg) reduced VO(2) by 80%, decreased PCr by 35%, moderately increased adenosine and lactate release, and progressively reduced DeltaL by 50% (to 5.6 +/- 3.3%). All parameters fully recovered during reoxygenation. PO(2)-dependent downregulation of DeltaL was accompanied by a progressive reduction in systolic [Ca(2+)](i) (from 512 +/- 110 to 357 +/- 91 nmol/l at 6 mmHg and to 251 +/- 69 nmol/l at 3 mmHg), whereas diastolic free [Ca(2+)](i) remained unchanged. Therefore, the mechanism of the reversible, PO(2)-dependent downregulation of contractile activity (myocardial hibernation) involves a substantial reduction of systolic calcium.     

Distinct signaling properties of mitogen-activated protein kinase kinases 4 (MKK4) and 7 (MKK7) in embryonic stem cell (ESC) differentiation

Signal transduction pathways are integral components of the developmental regulatory network that guides progressive cell fate determination. MKK4 and MKK7 are upstream kinases of the mitogen-activated protein kinases (MAPKs), responsible for channeling physiological and environmental signals to their cellular responses. Both kinases are essential for survival of mouse embryos, but because of embryonic lethality, their precise developmental roles remain largely unknown. Using gene knock-out mouse ESCs, we studied the roles of MKK4 and MKK7 in differentiation in vitro. While MKK4 and MKK7 were dispensable for ESC self-renewal and pluripotency maintenance, they exhibited unique signaling and functional properties in differentiation. MKK4 and MKK7 complemented each other in activation of the JNK-c-Jun cascades and loss of both led to senescence upon cell differentiation. On the other hand, MKK4 and MKK7 had opposite effects on activation of the p38 cascades during differentiation. Specifically, MKK7 reduced p38 activation, while Mkk7(-/-) ESCs had elevated phosphorylation of MKK4, p38, and ATF2, and increased MEF2C expression. Consequently, Mkk7(-/-) ESCs had higher expression of MHC and MLC and enhanced formation of contractile cardiomyocytes. In contrast, MKK4 was required for p38 activation and Mkk4(-/-) ESCs exhibited diminished p-ATF2 and MEF2C expression, resulting in impaired MHC induction and defective cardiomyocyte differentiation. Exogenous MKK4 expression partially restored the ability of Mkk4(-/-) ESCs to differentiate into cardiomyocytes. Our results uncover complementary and interdependent roles of MKK4 and MKK7 in development, and identify the essential requirement for MKK4 in p38 activation and cardiomyocyte differentiation.     

IFNbeta induction by influenza A virus is mediated by RIG-I which is regulated by the viral NS1 protein

Influenza A virus causes epidemics of respiratory diseases in humans leading to thousands of death annually. One of its major virulence factors, the non-structural protein 1 (NS1), exhibits interferon-antagonistic properties. While epithelial cells of the respiratory tract are the primary targets of influenza virus, the virus-sensing mechanisms in these cells eventually leading to IFNbeta production are incompletely understood. Here we show that infection of epithelial cells with NS1-deficient influenza A virus upregulated expression of two molecules that have been previously implicated in sensing of RNA viruses, the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5). Gene silencing and overexpression experiments demonstrated that RIG-I, its adapter interferon-beta promoter stimulator 1 (IPS-1) and interferon-regulated factor 3 (IRF3) were involved in influenza A virus-mediated production of the antiviral IFNbeta. In addition, we showed that the NS1 protein is capable to inhibit the RIG-I-induced signalling, a mechanism which corresponded to the observation that only NS1-deficient but not the wild-type virus induced high-level production of IFNbeta. In conclusion, we demonstrated a critical involvement of RIG-I, IPS-1 and IRF3 in influenza A virus infection of epithelial cells.     

Molecular interference of Cd(2+) with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd(2+) is known to exchange, with high affinity in a slow reaction, for the Ca(2+) cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd(2+) binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd(2+)-binding to those sites, we have studied how Cd(2+) affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd(2+) with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd(2+) were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y(Z) to P(680)(+) was inhibited; (ii) Q(A)(-) to Q(B) electron transfer was slowed down; (iii) the S(2) state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q(A)(-)Fe(2+) was modified but its amplitude was not sensitive to the presence of Cd(2+). In addition, the presence of both Ca(2+) and DCMU abolished Cd(2+)-induced effects partially and in different sites. The number of sites for Cd(2+) binding and the possible nature of these sites are discussed.     

Shear stress-induced c-fos activation is mediated by Rho in a calcium-dependent manner

We aimed at elucidating the molecular basis of c-fos promoter activation in vascular endothelial cells (ECs) in response to shear stress, with emphases on Rho family GTPases (Rho, Cdc42, and Rac) and intracellular calcium. Dominant-negative and constitutively activated mutants of these GTPases were used to block the action of upstream signals and to activate the downstream pathways, respectively. The role of intracellular calcium was assessed with intracellular calcium chelators. Only Rho, but not Cdc42 or Rac, is involved in the shear stress induction of c-fos. This Rho-mediated shear-induction of c-fos is dependent on intracellular calcium, but not on the Rho effector p160ROCK or actin filaments. While the inhibition of p160ROCK and its ensuing disruption of actin filaments decreased the basal c-fos activity in static ECs (no flow), it did not affect the shear-inductive effect. The calcium chelator BAPTA-AM inhibits the shear-induction, as well as the static level, of c-fos activity.