Two mechanisms of caspase 9 processing in double-stranded RNA- and virus-triggered apoptosis.

Viral double-stranded RNA (dsRNA) is a ubiquitous intracellular "alert signal" used by cells to detect viral infection and to mount anti-viral responses. DsRNA triggers a rapid (complete within 2-4 h) apoptosis in the highly-susceptible HeLa cell line. Here, we demonstrate that the apical event in this apoptotic cascade is the activation of procaspase 8. Downstream of caspase 8, the apoptotic signaling cascade bifurcates into a mitochondria-independent caspase 8/caspase 3 arm and a mitochondria-dependent, caspase 8/Bid/Bax/Bak/cytochrome c arm. Both arms impinge upon, and activate, procaspase 9 via two different cleavage sites within the procaspase 9 molecule (D330 and D315, respectively). This is the first in vivo demonstration that the "effector" caspase 3 plays an "initiator" role in the regulation of caspase 9. The dsRNA-induced apoptosis is potentiated by the inhibition of protein synthesis, whose role is to accelerate the execution of all apoptosis steps downstream of, and including, the activation of caspase 8. Thus, efficient apoptosis in response to viral dsRNA results from the co-operation of the two major apical caspases (8 and 9) and the dsRNA-activated protein kinase R (PKR)/ribonuclease L (RNase L) system that is essential for the inhibition of protein synthesis in response to viral infection.

Recruitment of TRADD, FADD, and caspase 8 to double-stranded RNA-triggered death inducing signaling complexes (dsRNA-DISCs).

Rapid elimination of virus-infected cells by apoptosis is an efficient anti-viral strategy. Double-stranded RNA (dsRNA), a viral product, is potently and rapidly apoptogenic in susceptible cells. Caspase 8 plays an important role in the dsRNA-induced apoptosis; however, the mechanisms of caspase 8 activation in response to dsRNA are unknown. We demonstrate here that, in HeLa cells, the dsRNA-triggered activation of caspase 8 is independent of ongoing proteins synthesis (and is, therefore, independent of changes in pro- and anti-apoptotic gene expression) and involves the formation of multiprotein dsRNA-triggered death inducing signaling complexes (dsRNA-DISCs). DsRNA-DISCs contain FADD, TRADD, and caspase 8; however, several experimental approaches suggest that death ligands and death receptors (such as Fas/Apo1 and DR4/Apo2) are not involved in the formation of dsRNA-DISCs.

Specifically targeting the CD22 receptor of human B-cell lymphomas with RNA damaging agents.

Targeting CD22 on human B-cells with a monoclonal antibody conjugated to a cytotoxic RNAse causes potent and specific killing of the lymphoma cells in vitro. This translates to anti-tumor effects in human lymphoma models in SCID mice. RNA damage caused by RNAses could be an important alternative to standard DNA damaging chemotherapeutics. Moreover, targeted RNAses may overcome problems of toxicity and immunogenicity associated with plant or bacterial toxin containing immunotoxins.

Activation of NF-kappaB by double-stranded RNA (dsRNA) in the absence of protein kinase R and RNase L demonstrates the existence of two separate dsRNA-triggered antiviral programs.

Double-stranded RNA (dsRNA) of viral origin triggers two programs of the innate immunity in virus-infected cells. One is intended to decrease the rate of host cell protein synthesis and thus to prevent viral replication. This program is mediated by protein kinase R (PKR) and by RNase L and contributes, eventually, to the self-elimination of the infected cell via apoptosis. The second program is responsible for the production of antiviral (type I) interferons and other alarmone cytokines and serves the purpose of preparing naive cells for the viral invasion. This second program requires the survival of the infected cell and depends on the expression of antiapoptotic genes through the activation of the NF-kappaB transcription factor. The second program therefore relies on ongoing transcription and translation. It has been proposed that PKR plays an essential role in the activation of NF-kappaB by dsRNA. Here we present evidence that the dsRNA-induced NF-kappaB activity and the expression of beta interferon and inflammatory cytokines do not require either PKR or RNase L. Our results indicate, therefore, that the two dsRNA-activated programs are separate and can function independently of each other.

Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis.

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.

Differential requirement for the stress-activated protein kinase/c-Jun NH(2)-terminal kinase in RNAdamage-induced apoptosis in primary and in immortalized fibroblasts.

Onconase, an anticancer ribonuclease, damages cellular tRNA and causes caspase-dependent apoptosis in targeted cells (M. S. Iordanov, O. P. Ryabinina, J. Wong, T. H. Dinh, D. L. Newton, S. M. Rybak, and B. E. Magun. Cancer Res. 60, 1983-1994, 2000). The proapoptotic action of onconase depends on its RNase activity, but the molecular mechanisms leading to RNA damage-induced caspase activation are completely unknown. In this study, we have investigated whether onconase activates two signal-transduction pathways commonly stimulated by conventional chemo- and radiotherapy, namely the stress-activated protein kinase (SAPK) cascade and the pathway leading to the activation of nuclear factor-kappa B (NF-kappaB). We found that, in all cell types tested, onconase is a potent activator of SAPK1 (JNK1 and JNK2) and SAPK2 (p38 MAP kinase), but that it is incapable of activating NF-kappaB. Inhibition of p38 MAP kinase activity with a pharmacological inhibitor, SB203580, demonstrated that p38 MAP kinase is not required for onconase cytotoxicity. Using explanted fibroblasts from mice that contain targeted disruption of both jnk1 and jnk2 alleles, we found that JNKs are important mediators of onconase-induced cytotoxicity. Surprisingly, following the immortalization of these same cells with human papilloma virus (HPV16) gene products E6 and E7, additional proapoptotic pathways (exclusive of JNK) were provoked by onconase. Our results demonstrate that onconase may activate proapoptotic pathways in tumor cells that are not able to be accessed in normal cells. These results present the possibility that the cytotoxic activity of onconase in normal cells may be reduced by blocking the activity of JNKs.

Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways.

Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH(2)-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i. e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.

Different mechanisms of c-Jun NH(2)-terminal kinase-1 (JNK1) activation by ultraviolet-B radiation and by oxidative stressors.

Irradiation of mammalian cells with ultraviolet-B radiation (UV-B) triggers the activation of a group of stress-activated protein kinases known as c-Jun NH(2)-terminal kinases (JNKs). UV-B activates JNKs via UV-B-induced ribotoxic stress. Because oxidative stress also activates JNKs, we have addressed the question of whether the ribotoxic and the oxidative stress responses are mechanistically similar. The pro-oxidants sodium arsenite, cadmium chloride, and hydrogen peroxide activated JNK1 with slow kinetics, whereas UV-B potentiated the activity of JNK1 rapidly. N-acetyl cysteine (a scavenger of reactive oxygen intermediates) abolished the ability of all oxidative stressors tested to activate JNK1, but failed to affect the activation of JNK1 by UV-B or by another ribotoxic stressor, the antibiotic anisomycin. In contrast, emetine, an inhibitor of the ribotoxic stress response, was unable to inhibit the activation of JNK1 by oxidative stressors. Although UV-A and long wavelength UV-B are the spectral components of the ultraviolet solar radiation that cause significant oxidative damage to macromolecules, the use of a filter to eliminate the radiation output from wavelengths below 310 nm abolished the activation of JNK1 by UV. Our results are consistent with the notion that UV-B and oxidative stressors trigger the activation of JNK1 through different signal transduction pathways.

The p38MAPK inhibitor SB203580 alleviates ultraviolet-induced phosphorylation at serine 389 but not serine 15 and activation of p53.

Phosphorylation of p53 at serine 389 has been shown to be responsive uniquely to UV but not gamma irradiation. This report describes identification of the UV-responsive p38MAPK protein as a serine 389 kinase. The immunoprecipitated p38MAPK from UV-irradiated murine embryonic testicular carcinoma F9 cells phosphorylated the serine 392 residue but not serine 15 of the human p53 protein in vitro and this phosphorylation was inhibited by a p38MAPK-specific chemical inhibitor SB203580. The inhibitor also remarkably alleviated the UV-caused induction and serine 389 but not serine 15 phosphorylation of the murine p53 protein in vivo. Subsequently, this compound suppressed transcriptional activity of p53 and partially retarded UV-induced apoptosis. Moreover, p53 bound to p38 as revealed by immunoprecipitation with anti-p53 antibodies from UV-treated F9 cells. Thus, these results suggest that UV-stimulated p53 phosphorylation at serine 389 is mediated by the stress-responsive p38MAPK.

Ultraviolet radiation triggers the ribotoxic stress response in mammalian cells.

The ribotoxic stress response, which is conserved between prokaryotes and eukaryotes, is a cellular reaction to cytotoxic interference with the function of the 3'-end of the large (23 S/28 S) ribosomal RNA. The 3'-end of the large rRNA is directly involved in the three sequential steps of translational elongation: the aminoacyl-tRNA binding, the peptidyl transfer, and the ribosomal translocation. In mammalian cells, the ribotoxic stress response involves activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase and the p38 mitogen-activated protein kinase and transcriptional induction of immediate early genes such as c-fos and c-jun. Active ribosomes are essential mediators of the ribotoxic stress response. We demonstrate here that the transcriptional response of mammalian cells to ultraviolet radiation (UV response) displays the characteristics of a ribotoxic stress response, inasmuch as (i) the activation of stress kinases and gene expression in response to UV requires the presence of active ribosomes at the moment of irradiation; (ii) UV irradiation inhibits protein synthesis; and (iii) irradiation of cells with UV causes specific damage to the 3'-end of the 28 S rRNA. In contrast, the activation of the stress kinases by hyperosmolarity, by the DNA-cross-linking agent diepoxybutane, or by growth factors and cytokines does not depend on the presence of active ribosomes. Our results identify UV as a potential ribotoxic stressor and support the notion that some of the cellular signaling cascades in response to UV might be generated in the ribosome, possibly triggered by damage to rRNA.

Loss of cellular K+ mimics ribotoxic stress. Inhibition of protein synthesis and activation of the stress kinases SEK1/MKK4, stress-activated protein kinase/c-Jun NH2-terminal kinase 1, and p38/HOG1 by palytoxin.

The tumor promoter palytoxin has been found to activate the stress-activated protein kinase/c-Jun NH2-terminal kinase 1 (SAPK/JNK1), and it also potentiates, as demonstrated here, the p38/HOG1 mitogen-activated protein kinase and the upstream activator of SAPK/JNK1, SEK1/MKK4. In search of possible mechanisms for both the cytotoxicity and the activation of stress kinases by palytoxin, we found that palytoxin is a potent inhibitor of cellular protein synthesis. The inhibition of translation by palytoxin does not result from its direct binding to the translational apparatus. We have previously demonstrated that ribotoxic stressors (Iordanov, M. S., Pribnow, D., Magun, J. L., Dinh, T.-H., Pearson, J. A., Chen, S. L.-Y., and Magun, B. E. (1997) Mol. Cell. Biol. 17, 3373-3381) signal the activation of SAPK/JNK1 by binding to or covalently modifying 28 S rRNA in ribosomes that are active at the time of exposure to the stressor. Palytoxin acted as a ribotoxic stressor, inasmuch as it required actively translating ribosomes at the time of exposure to activate SAPK/JNK1. Palytoxin has been shown to augment ion fluxes by binding to the Na+/K+-ATPase in the plasma membrane of cells. To determine whether altered fluxes of either Na+ or K+ could be responsible for the effects of palytoxin on translation and on activation of SAPK/JNK1, cells were exposed to palytoxin in modified culture medium in which a major portion of the Na+ was replaced by either K+ or by choline+. The substitution of Na+ by K+ strongly inhibited the ability of palytoxin both to inhibit protein translation and to activate SAPK/JNK1, whereas the substitution of Na+ by choline+ did not. These results suggest that palytoxin-induced efflux of cellular K+ mimics ribotoxic stress by provoking both translational inhibition and activation of protein kinases associated with cellular defense against stress.

Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA.

Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH2-terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and alpha-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or alpha-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not.

Complex interactions with direct repeats of a mitogen-responsive VL30 enhancer.

The RVL-3 VL30 enhancer is an LTR-derived triple direct repeat of 35 base pairs that mediates gene induction in response to several different intracellular signaling pathways. Using mobility shift assays, methylation interference and DNase I footprinting, we have investigated the physical interactions between the RVL-3 enhancer and components of nuclear extracts from Rat-1 cells. Each enhancer repeat unit contains a single binding site. Our studies suggest that binding to the double or triple repeat enhancer is cooperative, involving simultaneous occupation of two sites, with a preference for adjacent sites. Binding cooperativity would have implications for the mechanism of gene activation directed from the native VL30 enhancer.

Transient inhibition of protein synthesis induces the immediate early gene VL30: alternative mechanism for thapsigargin-induced gene expression.

Induction of gene expression in response to calcium ionophores or thapsigargin, which inhibits the calcium-ATPase responsible for sequestering intracellular calcium, has frequently been attributed to direct stimulatory events subsequent to the elevation of intracellular free calcium. VL30 is a murine gene that is transcriptionally induced in response to a large array of mitogenic and transforming stimuli. We have shown previously that an enhancer element within the VL30 promoter region is dependent upon cotreatment with thapsigargin or calcium ionophore for a full-scale induction of gene expression. In this report, we demonstrate that both thapsigargin and calcium ionophores induce a transient inhibition of protein synthesis in Rat-1 cells transfected with a VL30 enhancer-driven reporter construct. Recovery of protein synthesis is facilitated by cotreatment with epidermal growth factor or phorbol esters. Furthermore, treatment with cycloheximide or DTT, which inhibit protein synthesis without altering intracellular calcium levels, can substitute for thapsigargin or ionophores in stimulating VL30 gene expression. These results suggest that the stimulatory effects of thapsigargin and calcium ionophores on VL30 expression may be mediated, at least in part, by the ability of these agents to initiate stress responses associated with inhibition of protein synthesis.

Characterization of a unique enhancer element responsive to cyclic adenosine 3',5'-monophosphate and elevated calcium.

The VL30 family of defective retrovirus-like elements is abundantly transcribed in response to numerous transforming and proliferative stimuli. We have identified a novel enhancer element in the long terminal repeat of the transcriptionally active VL30 element RVL-3. The RVL-3 enhancer mediates a calcium-dependent induction of gene expression in response to treatment with either epidermal growth factor or the phorbol ester tumor promoter 12-O-tetradecanoylphorbol acetate. In this report we present in vivo and in vitro evidence indicating that the RVL-3 enhancer is also responsive to cAMP in the presence of elevated intracellular calcium. Proteins present in nuclear extracts obtained from Rat-1 fibroblasts bind specifically to a 20-basepair sequence within the RVL3 triple repeat. Competition binding studies and mutational analyses indicate that the cAMP responsiveness maps to the same region responsible for mediating the inductive response to epidermal growth factor and 12-O-tetradecanoylphorbol. The responsive sequence is different from previously described enhancer elements. This novel enhancer mediates transcription by multiple agonists and promotes a greater than additive increase in gene expression when more than one signal transduction pathway is stimulated simultaneously.

Endothelin induces transcription of fos/jun family genes: a prominent role for calcium ion.

The 21-amino acid mammalian peptide endothelin (ET) is a powerful vasoconstrictor, a mitogen for fibroblasts and vascular smooth muscle cells, and a potent effector for numerous tissues. Through extracellular interaction with G protein-coupled transmembrane receptors, ET stimulates intracellular second messenger events that in turn activate immediate early gene transcription. Using Northern blot hybridization and nuclear run-on analyses, we examined the modulation of c-fos, fos-B, fra-1, c-jun, and jun-B gene transcripts in Rat-1 fibroblasts after ET treatment. Furthermore, we investigated the role that intracellular Ca2+ transients played in effecting this gene regulation, using the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to block Ca(2+)-dependent transcription. Our results demonstrate that ET rapidly effects increased RNA levels for all five fos/jun family genes investigated, at least two of them by increasing gene transcription. Furthermore, our results argue that increased intracellular free Ca2+ is directly involved in the induction of these fos/jun family genes by ET. While mobilization of intracellular Ca2+ is not the only pathway to fos/jun gene induction used by ET, it is clearly a major component of the signaling apparatus that is set in motion by this potent effector.

Identification of a novel enhancer element mediating calcium-dependent induction of gene expression in response to either epidermal growth factor or activation of protein kinase C.

The VL30 family of defective murine retroviruses consists of 100 to 200 members, of which fewer than 5% appear to be transcriptionally active. A genomic clone of the transcriptionally active VL30 element RVL-3 was identified and sequenced. Genetic analysis indicated that a triple-repeat sequence within the RVL-3 long terminal repeat is capable of functioning as an inducible enhancer element responding to a variety of agonists. In Rat-1 fibroblasts, the ability of the RVL-3 enhancer to mediate induction of gene expression from a heterologous promoter in response to either epidermal growth factor (EGF) or phorbol ester treatment required coelevation of intracellular calcium. Two CArG boxes present in the triple-repeat sequence appeared to exert a negative effect on gene expression, as mutation of these sequences elevated the basal level of expression observed without altering the fold induction in response to either EGF or protein kinase C activation. In the presence of these CArG elements, mutation of AP-1-like sites adjacent to the CArG elements significantly inhibited the ability of either EGF or phorbol esters to induce gene expression. The effect of mutating these AP-1-like sites was relieved by simultaneous mutation of the CArG sites, indicating that interactions among these sites modulate RVL-3 expression. Mutational analysis and gel mobility shift experiments have identified a third sequence within the VL30 triple-repeat element that is required for the induction of gene expression and serves as a binding site for nuclear proteins. Sequence comparisons indicate that this enhancer element has not been described previously.

Regulation of transin/stromelysin and VL30 gene expression by intracellular calcium.

Changes in intracellular Ca++ levels are observed as a second messenger in response to a number of cellular agonists, including epidermal growth factor, transforming growth factor beta 1, and endothelin-1. The role of elevated intracellular Ca++ in transducing the effects of these three agonists on gene expression has been studied using two target genes: transin/stromelysin-1 and the endogenous murine retrovirus VL30. Although the effects of EGF and TGF beta 1 on transin/stromelysin-1 mRNA expression appear to be independent of these agonists' effects on intracellular Ca++ levels, elevated Ca++ interacted synergistically with activators of pkC to induce transin expression, even though neither agent alone could induce transin/stromelysin-1 expression. In contrast, the integrated VL30 retrovirus could be induced by Ca++ ionophores alone, and induction of VL30 mRNA by other agonists was blocked if intracellular Ca++ levels were held below a threshold value of 165 nM with Ca++ chelators. Genetic analysis of the VL30 upstream regulatory region indicated that a triple-repeat element present in the VL30 long-terminal repeat could function as an inducible enhancer, but responsiveness to either EGF or pkC activation required the concomitant elevation of intracellular Ca++. Because EGF was capable of inducing expression even in pkC-depleted cells, providing Ca++ levels were elevated, these results indicate that elevated intracellular Ca++ is capable of interacting synergistically with multiple signaling pathways to stimulate increased gene expression.

The role of C-Fos in growth factor regulation of stromelysin/transin gene expression.

Expression of the rat stromelysin (transin) gene is stimulated by growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), and inhibited by transforming growth factor-beta (TGF beta). Stimulation by both EGF and PDGF requires the presence of factors that recognize the AP-1 binding site in the stromelysin promoter, but PDGF stimulation requires induction of the protooncogene c-fos, while EGF acts through a FOS-independent pathway. The FOS-independent pathway appears to involve protein kinase C (PKC), since EGF, but not PDGF, requires activated protein kinase C to stimulate stromelysin expression. TGF beta inhibition of stromelysin gene expression requires an upstream sequence, referred to as the TGF beta inhibitory element (TIE). FOS is also a part of a protein complex that binds to the TIE. The protooncogene FOS is therefore involved in both stimulation and inhibition of stromelysin gene expression.

Stimulation of Ca2+ influx by endothelin-1 is subject to negative feedback by elevated intracellular Ca2+.

Endothelin-1 (ET-1) has been shown to require Ca2+ influx for activation of vascular smooth muscle in vivo, but in vitro models show that ET-1 mobilizes intracellular Ca2+ and is independent of extracellular Ca2+. We present data that suggest ET-1 modulates cellular responses through a dual mechanism involving both phosphatidylinositol turnover and Ca2+ channel activation. Addition of low concentrations of ET-1 (less than 10(-9) M) to serum-deprived quiescent Rat-1 cells stimulated Ca2+ influx while having little effect on diacylglycerol (DG) release or intracellular Ca2+ levels. In contrast, higher concentrations of ET-1 (greater than 10(-9) M) stimulated intracellular Ca2+ transients and release of inositol trisphosphate (IP3) and DG but did not activate Ca2+ uptake. Stimulation of Ca2+ influx at low [ET-1] could not be accounted for by depletion of intracellular IP3-sensitive pools. Neither the stimulation of Ca2+ influx at low [ET-1] nor the inhibitory actions of high [ET-1] could be mimicked by the activation of protein kinase C. We tested the hypothesis that elevated intracellular Ca2+ was inhibitory for Ca2+ influx. When intracellular Ca2+ transients were maintained below approximately 165 nM by chelation with BAPTA or BAPTA derivatives with altered affinity for Ca2+, Ca2+ influx was stimulated over the entire range of ET-1 concentrations. In addition, experimentally elevating intracellular Ca2+ levels with the tumor promoter thapsigargin abolished ET-1-stimulated Ca2+ influx. These data suggest that the biological consequences of ET-1 release may be determined by local concentration differences. Thus in vascular smooth muscle cells ET-1 may act either to mobilize intracellular Ca2+ or to promote Ca2+ influx, depending on the distance from the endothelial cell source in the vascular wall. The activation of different processes by low and high ET-1 concentrations may determine the physiological response to ET-1 stimulation in vivo.