Selective down-regulation of IP3receptor subtypes by caspases and calpain during TNF α -induced apoptosis of human T-lymphoma cells

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP₃R) [IP₃-gated Ca²⁺channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP₃R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca²⁺channel properties in an IP₃-dependent manner. We have also demonstrated that TNFα promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca²⁺response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFα, several IP₃R subtype-related changes occur (e.g. proteolysis of IP₃R subtypes, inhibition of IP₃binding and impairment of IP₃-mediated Ca²⁺flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFα-mediated perturbation of IP₃R1 and IP₃R2 (but not IP₃R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFα on IP₃R3 function. These findings suggest that IP₃R1 and IP₃R2 serve as cellular substrates for caspases, and IP₃R3 is a substrate for calpain. We propose that the selective down-regulation of IP₃R subtype-mediated Ca²⁺function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFα in human T-cells.     

Poliovirus protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface

Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-kappaB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.     

Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals

Intracellular calcium signals mediated by IP(3)and ryanodine receptors (IP(3)R/RyR) play a central role in cell survival, but emerging evidence suggests that IP(3)R/RyR are also important in apoptotic cell death. Switch from the life program to the death program may involve coincident detection of proapoptotic stimuli and calcium signals or changes in the spatiotemporal pattern of the calcium signal or changes at the level of effectors activated by the calcium signal (e.g. calpain, calcineurin). The fate of the cell is often determined in the mitochondria, where calcium spikes may support cell survival through stimulation of ATP production or initiate apoptosis v ia opening of the permeability transition pore and release of apoptotic factors such as cytochrome c. The functional importance of these mitochondrial calcium signalling pathways has been underscored by the elucidation of a highly effective, local Ca(2+)coupling between IP(3)R/RyR and mitochondrial Ca(2+)uptake sites. This article will focus on the IP(3)R/RyR-dependent pathways to apoptosis, particularly on the mitochondrial phase of the death cascade.     

Death receptor 3 mediates TNFSF15- and TNFα-induced endothelial cell apoptosis

Tumor necrosis factor superfamily 15 (TNFSF15) suppresses angiogenesis by specifically inducing apoptosis in proliferating endothelial cells. Death receptor 3 (DR3), a member of the TNF receptor superfamily (TNFRSF25), has been identified as a receptor for TNFSF15 to activate T cells. It is unclear, however, whether DR3 mediates TNFSF15 activity on endothelial cells. Here we show that siRNA-mediated knockdown of DR3 in an in vivo Matrigel angiogenesis assay, or in adult bovine aortic endothelial (ABAE) cell cultures, leads to resistance of endothelial cells to TNFSF15-induced apoptosis. Interestingly, DR3-depleted cells also exhibited markedly diminished responsiveness to TNFα cytotoxicity, even though DR3 is not a receptor for TNFα. Treatment of the cells with either TNFSF15 siRNA or a TNFSF15-neutralizing antibody, 4-3H, also results in a significant inhibition of TNFα-induced apoptosis. Mechanistically, DR3 siRNA treatment gives rise to an increase of ERK1/2 MAPK activity, and up-regulation of the anti-apoptotic proteins c-FLIP and Bcl-2, thus strengthening apoptosis-resisting potential in the cells. These findings indicate that DR3 mediates TNFSF15-induced endothelial cell apoptosis, and that up-regulation of TNFSF15 expression stimulated by TNFα is partly but significantly responsible for TNFα-induced apoptosis in endothelial cells.     

IP(3)-induced tension and IP(3)-receptor expression in rat soleus muscle during postnatal development

The present study was designed to examine whether changes in Ca(2+) release by inositol-1,4,5-trisphosphate (IP(3)) in 8-, 15-, and 30-day-old rat skeletal muscles could be associated with the expression of IP(3) receptors. Experiments were conducted in slow-twitch muscle in which both IP(3)-induced Ca(2+) release and IP(3)-receptor (IP(3)R) expression have been shown to be larger than in fast-twitch muscle. In saponin-skinned fibers, IP(3) induced transient contractile responses in which the amplitude was dependent on the Ca(2+)-loading period with the maximal IP(3) contracture being at 20 min of loading. The IP(3) tension decreased during postnatal development, was partially inhibited by ryanodine (100 microM), and was blocked by heparin (20-400 microg/ml). Amplification of the DNA sequence encoding for IP(3)R isoforms (using the RT-PCR technique) showed that in slow-twitch muscle, the type 2 isoform is mainly expressed, and its level decreases during postnatal development in parallel with changes in IP(3) responses in immature fibers. IP(3)-induced Ca(2+) release would then have greater participation in excitation-contraction coupling in developing fibers than in mature muscle.     

Selective reconstitution of human D4 dopamine receptor variants with Gi alpha subtypes

G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.     

IP3-induced cytosolic and nuclear Ca2+ signals in HL-1 atrial myocytes: Possible role of IP3 receptor subtypes

HL-1 cells are the adult cardiac cell lines available that continuously divide while maintaining an atrial phenotype. Here we examined the expression and localization of inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes, and investigated how pattern of IP₃-induced subcellular local Ca²⁺ signaling is encoded by multiple IP₃R subtypes in HL-1 cells. The type 1 IP₃R (IP₃R1) was expressed in the perinucleus with a diffuse pattern and the type 2 IP₃R (IP₃R2) was expressed in the cytosol with a punctate distribution. Extracellular ATP (1 mM) elicited transient intracellular Ca²⁺ releases accompanied by a Ca²⁺ oscillation, which was eliminated by the blocker of IP₃Rs, 2-APB, and attenuated by ryanodine. Direct introduction of IP₃ into the permeabilized cells induced Ca²⁺ transients with Ca²⁺ oscillations at ⩾ 20 μM of IP₃, which was removed by the inhibition of IP₃Rs using 2-APB and heparin. IP₃-induced local Ca²⁺ transients contained two distinct time courses: a rapid oscillation and a monophasic Ca²⁺ transient. The magnitude of Ca²⁺ oscillation was significantly larger in the cytosol than in the nucleus, while the monophasic Ca²⁺ transient was more pronounced in the nucleus. These results provide evidence for the molecular and functional expression of IP₃R1 and IP₃R2 in HL-1 cells, and suggest that such distinct local Ca²⁺ signaling may be correlated with the punctate distribution of IP₃R2s in the cytosol and the diffuse localization of IP₃R1 in the peri-nucleus.     

Activation of caspases and serine proteases during apoptosis induced by onconase (Ranpirnase)

Onconase (ONC) is a ribonuclease isolated from amphibian oocytes that is cytostatic and cytotoxic to numerous tumor lines. ONC shows in vivo anti-tumor activity in mouse tumor models and is currently in Phase III clinical trials. Previous studies indicated that ONC induces apoptosis of the target cells most likely along the mitochondrial pathway involving caspase-9 as the initiator caspase. We have recently developed an approach to detect the activation of serine (Ser) proteases during apoptosis. The method is based on affinity labeling of Ser protease active centers with fluorochrome-tagged inhibitors. The aim of the present study was to reveal whether Ser proteases are activated during apoptosis induced by ONC. Human leukemic HL-60 cells were treated with ONC for up to 72 h and then exposed to 5(6)-carboxyfluoresceinyl-L-phenylalanylchloromethyl ketone (FFCK) or 5(6)-carboxyfluoresceinyl-L-leucylchloromethyl ketone (FLCK), the fluorescing green reagents reactive with active centers of the chymotrypsin-like enzymes that cleave proteins at the Phe (FFCK) or Leu (FLCK) site. Activation of caspases was assayed in the same cells using sulforhodamine-labeled (fluorescing red) pan-caspases inhibitor (SR-VAD-FMK). Administration of 1.67 microM ONC into cultures of HL-60 cells led to the appearance of cells that bound SR-VAD-FMK as well as FFCK and FLCK. Most labeled cells had features characteristic of apoptosis. We interpret the binding of these ligands, which was irreversible and withstood cell fixation, as revealing activation of caspases and chymotrypsin-like Ser proteases. Because the induction of binding of each of the three ligands occurred at approximately the same time, the data suggest that during apoptosis caspases and Ser proteases may transactivate each other. The intercellular and subcellular pattern of binding SR-VAD-FMK vs FFCK or vs FLCK was different indicating a variability in abundance and localization of these enzymes within individual apoptotic cells. The FFCK- and FLCK-reactive proteins were of similar molecular mass, approximately 59 and approximately 57 kDa, respectively.     

Characterization of inositol 1,4,5-trisphosphate (IP3) receptor subtypes at rat colonic epithelium

The aim of the present study was the characterization of the subtypes of inositol 1,4,5-trisphosphate receptors (IP3R) in rat colonic epithelium. A monoclonal antibody against IP3R1 did not stain the colonic epithelial cells. In contrast, IP3R2 and IP3R3 were found within the epithelium; however, with a distinct intracellular localization and differences in their distribution along the crypt axis. IP3R2 immunoreactivity was found within the nuclei of the epithelial cells. The signal was distributed all over the nucleus and not restricted to the nuclear envelope as demonstrated by counterstaining with lamin B1 and electron microscopical examination after immunogold labelling. In contrast, an antibody against IP3R3 stained the epithelial cells mostly in their apical half in accordance with the typical localization of IP3R in organelles such as the endoplasmic reticulum. In addition, there was a gradient from the surface region towards the crypt fundus, where the IP3R3 signal could not be detected. Despite the strong IP3R3-gradient, in saponin-permeabilized colonic crypts exogenously administered IP3 or adenophostin A evoked a similar depletion of mag-fura-2-loaded intracellular Ca2+ stores in crypt and surface cells suggesting a contribution of the nuclear IP3R2 to the Ca2+ release. This conclusion was confirmed by experiments with isolated nuclei from colonic epithelium, at which IP3 was able to induce changes in the Ca2+ concentration, which were inhibited by 2-aminoethoxy-diphenylborate (2-APB), a blocker of IP3 receptors. These results demonstrate that the colonic epithelial cells undergo changes in IP3R subtype expression during differentiation.     

Sequential activation of caspases and serine proteases (serpases) during apoptosis

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TICK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP-reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.     

IP(3)-mediated Ca(2+) signals in human neuroblastoma SH-SY5Y cells with exogenous overexpression of type 3 IP(3) receptor

Human neuroblastoma SH-SY5Y cells, predominantly expressing type 1 inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), were stably transfected with IP(3)R type 3 (IP(3)R3) cDNA. Immunocytochemistry experiments showed a homogeneous cytoplasmic distribution of type 3 IP(3)Rs in transfected and selected high expression cloned cells. Using confocal Ca(2+) imaging, carbachol (CCh)-induced Ca(2+) release signals were studied. Low CCh concentrations (< or = 750 nM) evoked baseline Ca(2+) oscillations. Transfected cells displayed a higher CCh responsiveness than control or cloned cells. Ca(2+) responses varied between fast, large Ca(2+) spikes and slow, small Ca(2+) humps, while in the clone only Ca(2+) humps were observed. Ca(2+) humps in the transfected cells were associated with a high expression level of IP(3)R3. At high CCh concentrations (10 microM) Ca(2+) transients in transfected and cloned cells were similar to those in control cells. In the clone exogenous IP(3)R3 lacked the C-terminal channel domain but IP(3)-binding capacity was preserved. Transfected cells mainly expressed intact type 3 IP(3)Rs but some protein degradation was also observed.We conclude that in transfected cells expression of functional type 3 IP(3)Rs causes an apparent higher affinity for IP(3). In the clone, the presence of degraded receptors leads to an efficient cellular IP(3) buffer and attenuated IP(3)-evoked Ca(2+) release.     

Cleavage of focal adhesion kinase by different proteases during SRC-regulated transformation and apoptosis. Distinct roles for calpain and caspases

Integrin-associated focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. In vitro, cells utilize a complex temporal and spatially regulated mechanism of focal adhesion assembly and disassembly required for cell migration. Recent studies indicate that members of both calpain and caspase protease families can promote limited proteolytic cleavage of several components of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such as tumor invasion. v-Src-induced oncogenic transformation is associated with loss of focal adhesion structures and transition to a less adherent, more motile phenotype, while inactivating temperature-sensitive v-Src in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that v-Src-induced disassembly of focal adhesions is accompanied by calpain-dependent proteolysis of focal adhesion kinase. Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion disruption, loss of substrate adhesion, and cell migration. In contrast, focal adhesion loss during detachment and apoptosis induced after switching off temperature-sensitive v-Src in serum-deprived transformed cells is accompanied by caspase-mediated proteolysis of focal adhesion kinase. Thus, calpain and caspase differentially regulate focal adhesion turnover during Src-regulated cell transformation, motility, and apoptosis.     

Effect of 1alpha,25(OH)2-vitamin D3 on TNF alpha-mediated apoptosis of human primary osteoblast-like cells in vitro.

1alpha,25(OH)2-vitamin D3 is a hormone which potentially stimulates bone cell growth and differentiation. TNFalpha is one possible inductor for apoptosis; apoptosis being an important regulatoring factor for bone modelling and remodelling. We examined the influence of physiological levels (0.1 nM) 1alpha,25(OH)2-vitamin D3 on TNFalpha-mediated apoptosis in human osteoblast-like cells. These human cells were obtained from bone fragments obtained during orthopedic operations on patients without systemic bone disease. Treatment with 1alpha,25(OH)2-vitamin D3 for 8 weeks resulted in a significant reduction (30%) of viable cell number compared to untreated cells. Incubation with TNFalpha (100 ng/ml for 4 hours) only had limited effects on the rate of apoptosis in control cells. After pretreatment with 1alpha,25(OH)2-vitamin D3, induction of apoptosis increased up to 10% in human osteoblast-like cells. In parallel to the induction of apoptosis, 1alpha,25(OH)2-vitamin D3 stimulated osteocalcin and alkaline phosphatase as markers of mature osteoblasts. Our data suggest that 1alpha,25(OH)2-vitamin D3 has a stimulatory effect on TNFalpha-induced apoptosis in human osteoblast-like cells as a result of 1alpha,25(OH)2-vitamin D3-induced cell differentiation.     

Proteolysis of the human DNA polymerase epsilon catalytic subunit by caspase-3 and calpain specifically during apoptosis

Human DNA polymerase epsilon (pol ) normally contains a 261-kDa catalytic subunit (p261), but from some sources it is isolated as a 140-kDa catalytic core of p261. This shortened form possesses normal or somewhat enhanced polymerase activity and its significance is unknown. We report here that caspase-3 and calpain can form p140 from p261 in vitro and in vivo and that during early stages of apoptosis induced in Jurkat cells by staurosporine or anti-Fas-activating antibody, p261 is cleaved into p140 by caspase-3. At later stages, activated calpain might also contribute to this conversion. The sites of cleavage by caspase-3 have been identified, and mutations at these ‘DEAD boxes’ resulted in cleavage-resistant enzyme. Cleavage at these sites separates the ‘N-terminal catalytic core’ from the ‘C-terminal’ regions described for p261. Cleavage does not occur during necrosis or following exposure to H₂O₂ or methanesulfonic acid methyl ester. p140 is unlikely to be able to functionally replace p261 in vivo, since it does not bind to PCNA or the other pol subunits.     

Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells.

We have previously reported that CD40 stimulation sensitizes human memory B cells to undergo apoptosis upon subsequent B cell receptor (BCR) ligation. We have proposed that activation stimuli connect the BCR to an apoptotic pathway in mature B cells and that BCR-induced apoptosis of activated B cells could serve a similar function as activation-induced cell death in the mature T cell compartment. Although it has been reported that caspases are activated during this process, the early molecular events that link the Ag receptor to these apoptosis effectors are largely unknown. In this study, we report that acquisition of susceptibility to BCR-induced apoptosis requires entry of memory B cells into the S phase of the cell cycle. We also show that transduction of the death signal via the BCR sequentially proceeds through a caspase-independent and a caspase-dependent phase, which take place upstream and downstream of the mitochondria, respectively. Furthermore, our data indicate that the BCR-induced alterations of the mitochondrial functions are involved in activation of the caspase cascade. We have found both caspases-3 and -9, but not caspase-8, to be involved in the BCR apoptotic pathway, thus supporting the notion that initiation of the caspase cascade could be under the control of the caspase-9/Apaf-1/cytochrome c multimolecular complex. Altogether, our findings establish the mitochondria as the connection point through which the Ag receptor can trigger the executioners of apoptotic cell death in mature B lymphocytes.