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Michael Y. Sherman - One of the best experts on this subject based on the ideXlab platform.
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Regulation of necrosis of H9c2 myogenic cells upon transient energy deprivation. Rapid deenergization of mitochondria precedes necrosis and is controlled by reactive oxygen species, Stress Kinase JNK, HSP72 and ARC.
The Journal of biological chemistry, 2003Co-Authors: Julia A. Yaglom, Vladimir L. Gabai, Daryoush Ekhterae, Michael Y. ShermanAbstract:Abstract Subjecting myogenic H9c2 cells to transient energy deprivation leads to a caspase-independent death with typical features of necrosis. Here we show that the rupture of cytoplasmic membrane, the terminal event in necrosis, is shortly preceded by rapid depolarization of mitochondrial membranes. The rapid deenergization of mitochondria critically depended upon prior generation of reactive oxygen species (ROS) during ATP depletion stage. Accordingly, expression of catalase prevented mitochondrial depolarization and averted subsequent necrosis. Interestingly, trifluoperazine, a compound that protects cells from ischemic insults, prevented necrosis of H9c2 cells through inhibition of ROS production. Other factors that regulated the mitochondrial membrane depolarization and subsequent loss of plasma membrane integrity include a Stress Kinase JNK activated at early steps of recovery from ATP depletion, as well as an apoptotic inhibitory protein ARC. Accordingly, inhibition of JNK or overexpression of ARC prevented mitochondrial depolarization and rescued H9c2 cells from necrosis. ROS and JNK affected mitochondrial deenergization and necrosis independently of each other since inhibition of ROS production did not prevent activation of JNK, whereas inhibition of JNK did not suppress ROS accumulation. Therefore, JNK activation and ROS production represent two independent pathways that control mitochondrial depolarization and subsequent necrosis of cells subjected to transient energy deprivation. Overexpression of ARC, although preventing mitochondrial depolarization, did not affect either JNK activation or production of ROS. The major heat shock protein Hsp72 inhibited JNK-related steps of necrotic pathway but did not affect ROS accumulation. Interestingly, mitochondrial depolarization and subsequent necrosis can be suppressed by an Hsp72 mutant Hsp72ΔEEVD, which lacks chaperone function but can efficiently suppress JNK activation. Thus, Hsp72 is directly implicated in a signaling pathway, which leads to necrotic death.
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Hsp72 and Stress Kinase c-jun N-Terminal Kinase Regulate the Bid-Dependent Pathway in Tumor Necrosis Factor-Induced Apoptosis
Molecular and cellular biology, 2002Co-Authors: Vladimir L. Gabai, Dick D. Mosser, Katsuhide Mabuchi, Michael Y. ShermanAbstract:The major inducible heat shock protein Hsp72 protects cells from a variety of Stressful conditions, including heat shock, ischemia, UV radiation, tumor necrosis factor (TNF), and anticancer drugs (for reviews see references 1, 13, and 17). A number of studies have recently demonstrated that in the protection of cells Hsp72 suppresses a Stress-induced apoptotic program, but the mechanism of Hsp72 action in apoptotic signal transduction is still a matter of debate. The key element of the apoptotic program is the efflux of cytochrome c from mitochondria to cytosol, where it subsequently forms a complex (apoptosome) with Apaf-1 and caspase 9, leading to activation of the latter (3). In turn, active caspase 9 cleaves and activates the major effector caspase, caspase 3, leading to the execution of apoptosis. A number of studies from several labs have demonstrated that Hsp72 blocks Stress-induced activation of the caspase cascade (2, 5, 12, 25, 27, 30) (although it should be noted that there was a report that Hsp72 can also regulate apoptosis downstream of caspases [18]). Experiments with cell lysates showed that Hsp72 may directly prevent apoptosome formation (2, 30) and caspase 3 activation (19). Other studies, however, indicated that in vivo Hsp72 functions at an early step, upstream of mitochondria and caspase activation. For instance, Hsp72 overexpression was shown to prevent cytochrome c release after heat shock (27) or hydrogen peroxide (7), although the mechanism of this effect of Hsp72 is unknown. It appears that Hsp72 can preserve mitochondrial integrity during activation of the apoptotic program by suppression of a signaling pathway that leads to mitochondrial damage (e.g., release of cytochrome c). Indeed, Hsp72 can block activation of a Stress Kinase, c-jun N-terminal Kinase (JNK) (12, 25), an indispensable early element in Stress-induced apoptotic program which controls the release of cytochrome c (34). In fact, it has recently been demonstrated that in murine embryonal fibroblasts derived from a JNK knockout mouse (a mutant with deletion of the two major isoforms JNK1 and JNK2), cytochrome c cannot be released from mitochondria after exposure to UV irradiation and some other Stressful treatments (8, 35). Hsp72 and JNK also appear to play a general role in the control of various caspase-independent types of cell death (14, 15). Hsp72 is a molecular chaperone involved in refolding and degradation of Stress-damaged proteins (see reference 9 for a review). It binds to denatured polypeptides via its peptide-binding domain and promotes their refolding in an ATP-dependent process (16). Deletion of the ATPase domain locks Hsp72 in a substrate-bound form which inhibits the refolding reaction (4). Whether the chaperone function of Hsp72 is required for its antiapoptotic activity has been a controversy. Several studies demonstrated that the protein refolding function of Hsp72 is dispensable for suppression of JNK activation and apoptosis, since the ATPase deletion mutant of Hsp72, CTF (C-terminal fragment), was fully active in protection of fibroblasts from heat- or UV-induced cell death (19, 20, 28, 37, 39). On the other hand, a recent study with lymphoid cells demonstrated that the chaperone function of Hsp72 is necessary for protection (27). In this work it was shown that normal Hsp72 or the CTF mutant could efficiently block activation of JNK by heat shock, but only normal Hsp72 could protect cells from heat-induced apoptosis (27). Especially interesting were the data with a mutant form of Hsp72 in which four critical C-terminal amino acids, EEVD, that are necessary for the chaperone function of Hsp72 were lacking or were replaced with AAAA (11). In contrast to normal Hsp72, this Hsp72 mutant could not prevent cytochrome c-induced caspase 3 activation in vitro (2) and heat-induced apoptosis in vivo (27); however, it efficiently inhibited JNK activity (27). Since specific inhibition of JNK was demonstrated to be sufficient to block heat-induced apoptosis in several cellular models (15, 36, 41), the data with these mutants appeared puzzling. These data could either suggest that Hsp72 can block apoptosis in a JNK-independent manner or that a latent JNK-independent pathway could be activated by heat shock when cells express Hsp72 mutants but not normal Hsp72. In other words, according to the latter interpretation, when the mutant form of Hsp72 is expressed, protein damage in cells exposed to heat shock is so severe that cells start to die in a JNK-independent manner. On the other hand, when normal Hsp72 is expressed, JNK is inhibited and proteins become repaired, leading to suppression of apoptosis. There were two goals of this study: (i) to elucidate whether the chaperone function of Hsp72 is necessary for inhibition of the apoptotic program initiated by agents that do not damage proteins and (ii) to clarify the effect of Hsp72 on the apoptotic program.
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Suppression of Stress Kinase JNK Is Involved in HSP72-mediated Protection of Myogenic Cells from Transient Energy Deprivation HSP72 ALLEVIATES THE Stress-INDUCED INHIBITION OF JNK DEPHOSPHORYLATION
The Journal of biological chemistry, 2000Co-Authors: Vladimir L. Gabai, Anatoli B. Meriin, Julia A. Yaglom, Jeanne Y. Wei, Dick D. Mosser, Michael Y. ShermanAbstract:Since protection of cells from Stress-induced apoptosis by the heat shock protein Hsp72 involves suppression of Stress Kinase JNK, we suggested that Hsp72-mediated JNK inhibition might also be critical for myocardial protection from ischemia/reperfusion. Transient energy deprivation of H9c2 myogenic cells, used as an in vitro model of myocardial ischemia, led to cell death that had morphological features of apoptosis and necrosis and was independent of caspases. Surprisingly, this unusual type of cell death was regulated by JNK and ERK Kinases. In fact, specific inhibition of JNK increased cell survival; specific inhibition of ERKs enhanced deleterious consequences of energy deprivation, whereas inhibition of p38 Kinase had no effect. Hsp72 suppressed activation of JNK and did not increase ERK activity, suggesting that inhibition of JNK is the important component of Hsp72-mediated protection. Upon transient energy deprivation, activation of JNK proceeds via two distinct pathways, stimulation of JNK phosphorylation by a protein Kinase SEK1 and inhibition of JNK dephosphorylation. Remarkably, in cells exposed to transient energy deprivation, Hsp72 enhanced the rate of JNK dephosphorylation but did not affect SEK1 activity. Therefore, it appears that Hsp72 specifically down-regulates JNK by accelerating its dephosphorylation, which reduces the susceptibility of cardiac cells to simulated ischemia/reperfusion.
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HSP72 can protect cells from heat-induced apoptosis by accelerating the inactivation of Stress Kinase JNK.
Cell stress & chaperones, 2000Co-Authors: Vladimir Volloch, Vladimir L. Gabai, Sophia Rits, Thomas Force, Michael Y. ShermanAbstract:The major heat shock protein Hsp72 prevents heat-induced apoptosis. We have previously demonstrated that transiently expressed Hsp72 exerts its anti-apoptotic effect by suppressing the activity of Stress-Kinase JNK, an early component of the apoptotic pathway initiated by heat shock. On the other hand, constitutive expression of Hsp72 does not lead to suppression of heat-induced JNK activation, yet still efficiently prevents apoptosis. To address this apparent contradiction, we studied the effects of constitutively expressed Hsp72 on activation of JNK and apoptosis in Rat-1 fibroblasts. We found that the level of heat-induced apoptosis directly correlated with the duration rather than the magnitude of JNK activity following heat shock. Constitutively expressed Hsp72 strongly reduced the duration of JNK while it did not suppress initial JNK activation. These effects were due to Hsp72-mediated acceleration of JNK dephosphorylation. Addition of vanadate to inhibit JNK phosphatase activity completely prevented the anti-apoptotic action of Hsp72. Therefore, suppression of heat-induced apoptosis by Hsp72 could be fully accounted for by its effects on JNK activity.
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Role of Hsp70 in regulation of Stress‐Kinase JNK: implications in apoptosis and aging
FEBS Letters, 1998Co-Authors: Vladimir L. Gabai, Anatoli B. Meriin, Julia A. Yaglom, Vladimir Volloch, Michael Y. ShermanAbstract:Cell protection from Stresses by the major heat shock protein Hsp72 was previously attributed to its ability to prevent aggregation and to accelerate refolding of damaged proteins. This repair function of Hsp72 may play an important role in cell survival after extremely harsh protein damaging treatments leading to necrotic cell death. On the other hand, protein repair function of Hsp72 cannot explain how it protects cells from Stresses which do not cause direct protein damage, e.g. some genotoxic agents. These Stresses kill cells through activation of apoptosis, and Hsp72 increases cell survival by interfering with the apoptotic program. Recently it has been found that Hsp72 mediates suppression of a Stress-activated protein Kinase, JNK, an early component of Stress-induced apoptotic signalling pathway. This finding provides the basis for the anti-apoptotic activity of Hsp72. These observations can explain increased Stress sensitivity of aged cells in which compromised inducibility of Hsp72 leads to a loss of control of JNK activation by Stresses and subsequently to a higher rate of apoptotic death.
Vladimir L. Gabai - One of the best experts on this subject based on the ideXlab platform.
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Regulation of necrosis of H9c2 myogenic cells upon transient energy deprivation. Rapid deenergization of mitochondria precedes necrosis and is controlled by reactive oxygen species, Stress Kinase JNK, HSP72 and ARC.
The Journal of biological chemistry, 2003Co-Authors: Julia A. Yaglom, Vladimir L. Gabai, Daryoush Ekhterae, Michael Y. ShermanAbstract:Abstract Subjecting myogenic H9c2 cells to transient energy deprivation leads to a caspase-independent death with typical features of necrosis. Here we show that the rupture of cytoplasmic membrane, the terminal event in necrosis, is shortly preceded by rapid depolarization of mitochondrial membranes. The rapid deenergization of mitochondria critically depended upon prior generation of reactive oxygen species (ROS) during ATP depletion stage. Accordingly, expression of catalase prevented mitochondrial depolarization and averted subsequent necrosis. Interestingly, trifluoperazine, a compound that protects cells from ischemic insults, prevented necrosis of H9c2 cells through inhibition of ROS production. Other factors that regulated the mitochondrial membrane depolarization and subsequent loss of plasma membrane integrity include a Stress Kinase JNK activated at early steps of recovery from ATP depletion, as well as an apoptotic inhibitory protein ARC. Accordingly, inhibition of JNK or overexpression of ARC prevented mitochondrial depolarization and rescued H9c2 cells from necrosis. ROS and JNK affected mitochondrial deenergization and necrosis independently of each other since inhibition of ROS production did not prevent activation of JNK, whereas inhibition of JNK did not suppress ROS accumulation. Therefore, JNK activation and ROS production represent two independent pathways that control mitochondrial depolarization and subsequent necrosis of cells subjected to transient energy deprivation. Overexpression of ARC, although preventing mitochondrial depolarization, did not affect either JNK activation or production of ROS. The major heat shock protein Hsp72 inhibited JNK-related steps of necrotic pathway but did not affect ROS accumulation. Interestingly, mitochondrial depolarization and subsequent necrosis can be suppressed by an Hsp72 mutant Hsp72ΔEEVD, which lacks chaperone function but can efficiently suppress JNK activation. Thus, Hsp72 is directly implicated in a signaling pathway, which leads to necrotic death.
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Hsp72 and Stress Kinase c-jun N-Terminal Kinase Regulate the Bid-Dependent Pathway in Tumor Necrosis Factor-Induced Apoptosis
Molecular and cellular biology, 2002Co-Authors: Vladimir L. Gabai, Dick D. Mosser, Katsuhide Mabuchi, Michael Y. ShermanAbstract:The major inducible heat shock protein Hsp72 protects cells from a variety of Stressful conditions, including heat shock, ischemia, UV radiation, tumor necrosis factor (TNF), and anticancer drugs (for reviews see references 1, 13, and 17). A number of studies have recently demonstrated that in the protection of cells Hsp72 suppresses a Stress-induced apoptotic program, but the mechanism of Hsp72 action in apoptotic signal transduction is still a matter of debate. The key element of the apoptotic program is the efflux of cytochrome c from mitochondria to cytosol, where it subsequently forms a complex (apoptosome) with Apaf-1 and caspase 9, leading to activation of the latter (3). In turn, active caspase 9 cleaves and activates the major effector caspase, caspase 3, leading to the execution of apoptosis. A number of studies from several labs have demonstrated that Hsp72 blocks Stress-induced activation of the caspase cascade (2, 5, 12, 25, 27, 30) (although it should be noted that there was a report that Hsp72 can also regulate apoptosis downstream of caspases [18]). Experiments with cell lysates showed that Hsp72 may directly prevent apoptosome formation (2, 30) and caspase 3 activation (19). Other studies, however, indicated that in vivo Hsp72 functions at an early step, upstream of mitochondria and caspase activation. For instance, Hsp72 overexpression was shown to prevent cytochrome c release after heat shock (27) or hydrogen peroxide (7), although the mechanism of this effect of Hsp72 is unknown. It appears that Hsp72 can preserve mitochondrial integrity during activation of the apoptotic program by suppression of a signaling pathway that leads to mitochondrial damage (e.g., release of cytochrome c). Indeed, Hsp72 can block activation of a Stress Kinase, c-jun N-terminal Kinase (JNK) (12, 25), an indispensable early element in Stress-induced apoptotic program which controls the release of cytochrome c (34). In fact, it has recently been demonstrated that in murine embryonal fibroblasts derived from a JNK knockout mouse (a mutant with deletion of the two major isoforms JNK1 and JNK2), cytochrome c cannot be released from mitochondria after exposure to UV irradiation and some other Stressful treatments (8, 35). Hsp72 and JNK also appear to play a general role in the control of various caspase-independent types of cell death (14, 15). Hsp72 is a molecular chaperone involved in refolding and degradation of Stress-damaged proteins (see reference 9 for a review). It binds to denatured polypeptides via its peptide-binding domain and promotes their refolding in an ATP-dependent process (16). Deletion of the ATPase domain locks Hsp72 in a substrate-bound form which inhibits the refolding reaction (4). Whether the chaperone function of Hsp72 is required for its antiapoptotic activity has been a controversy. Several studies demonstrated that the protein refolding function of Hsp72 is dispensable for suppression of JNK activation and apoptosis, since the ATPase deletion mutant of Hsp72, CTF (C-terminal fragment), was fully active in protection of fibroblasts from heat- or UV-induced cell death (19, 20, 28, 37, 39). On the other hand, a recent study with lymphoid cells demonstrated that the chaperone function of Hsp72 is necessary for protection (27). In this work it was shown that normal Hsp72 or the CTF mutant could efficiently block activation of JNK by heat shock, but only normal Hsp72 could protect cells from heat-induced apoptosis (27). Especially interesting were the data with a mutant form of Hsp72 in which four critical C-terminal amino acids, EEVD, that are necessary for the chaperone function of Hsp72 were lacking or were replaced with AAAA (11). In contrast to normal Hsp72, this Hsp72 mutant could not prevent cytochrome c-induced caspase 3 activation in vitro (2) and heat-induced apoptosis in vivo (27); however, it efficiently inhibited JNK activity (27). Since specific inhibition of JNK was demonstrated to be sufficient to block heat-induced apoptosis in several cellular models (15, 36, 41), the data with these mutants appeared puzzling. These data could either suggest that Hsp72 can block apoptosis in a JNK-independent manner or that a latent JNK-independent pathway could be activated by heat shock when cells express Hsp72 mutants but not normal Hsp72. In other words, according to the latter interpretation, when the mutant form of Hsp72 is expressed, protein damage in cells exposed to heat shock is so severe that cells start to die in a JNK-independent manner. On the other hand, when normal Hsp72 is expressed, JNK is inhibited and proteins become repaired, leading to suppression of apoptosis. There were two goals of this study: (i) to elucidate whether the chaperone function of Hsp72 is necessary for inhibition of the apoptotic program initiated by agents that do not damage proteins and (ii) to clarify the effect of Hsp72 on the apoptotic program.
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Suppression of Stress Kinase JNK Is Involved in HSP72-mediated Protection of Myogenic Cells from Transient Energy Deprivation HSP72 ALLEVIATES THE Stress-INDUCED INHIBITION OF JNK DEPHOSPHORYLATION
The Journal of biological chemistry, 2000Co-Authors: Vladimir L. Gabai, Anatoli B. Meriin, Julia A. Yaglom, Jeanne Y. Wei, Dick D. Mosser, Michael Y. ShermanAbstract:Since protection of cells from Stress-induced apoptosis by the heat shock protein Hsp72 involves suppression of Stress Kinase JNK, we suggested that Hsp72-mediated JNK inhibition might also be critical for myocardial protection from ischemia/reperfusion. Transient energy deprivation of H9c2 myogenic cells, used as an in vitro model of myocardial ischemia, led to cell death that had morphological features of apoptosis and necrosis and was independent of caspases. Surprisingly, this unusual type of cell death was regulated by JNK and ERK Kinases. In fact, specific inhibition of JNK increased cell survival; specific inhibition of ERKs enhanced deleterious consequences of energy deprivation, whereas inhibition of p38 Kinase had no effect. Hsp72 suppressed activation of JNK and did not increase ERK activity, suggesting that inhibition of JNK is the important component of Hsp72-mediated protection. Upon transient energy deprivation, activation of JNK proceeds via two distinct pathways, stimulation of JNK phosphorylation by a protein Kinase SEK1 and inhibition of JNK dephosphorylation. Remarkably, in cells exposed to transient energy deprivation, Hsp72 enhanced the rate of JNK dephosphorylation but did not affect SEK1 activity. Therefore, it appears that Hsp72 specifically down-regulates JNK by accelerating its dephosphorylation, which reduces the susceptibility of cardiac cells to simulated ischemia/reperfusion.
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HSP72 can protect cells from heat-induced apoptosis by accelerating the inactivation of Stress Kinase JNK.
Cell stress & chaperones, 2000Co-Authors: Vladimir Volloch, Vladimir L. Gabai, Sophia Rits, Thomas Force, Michael Y. ShermanAbstract:The major heat shock protein Hsp72 prevents heat-induced apoptosis. We have previously demonstrated that transiently expressed Hsp72 exerts its anti-apoptotic effect by suppressing the activity of Stress-Kinase JNK, an early component of the apoptotic pathway initiated by heat shock. On the other hand, constitutive expression of Hsp72 does not lead to suppression of heat-induced JNK activation, yet still efficiently prevents apoptosis. To address this apparent contradiction, we studied the effects of constitutively expressed Hsp72 on activation of JNK and apoptosis in Rat-1 fibroblasts. We found that the level of heat-induced apoptosis directly correlated with the duration rather than the magnitude of JNK activity following heat shock. Constitutively expressed Hsp72 strongly reduced the duration of JNK while it did not suppress initial JNK activation. These effects were due to Hsp72-mediated acceleration of JNK dephosphorylation. Addition of vanadate to inhibit JNK phosphatase activity completely prevented the anti-apoptotic action of Hsp72. Therefore, suppression of heat-induced apoptosis by Hsp72 could be fully accounted for by its effects on JNK activity.
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Role of Hsp70 in regulation of Stress‐Kinase JNK: implications in apoptosis and aging
FEBS Letters, 1998Co-Authors: Vladimir L. Gabai, Anatoli B. Meriin, Julia A. Yaglom, Vladimir Volloch, Michael Y. ShermanAbstract:Cell protection from Stresses by the major heat shock protein Hsp72 was previously attributed to its ability to prevent aggregation and to accelerate refolding of damaged proteins. This repair function of Hsp72 may play an important role in cell survival after extremely harsh protein damaging treatments leading to necrotic cell death. On the other hand, protein repair function of Hsp72 cannot explain how it protects cells from Stresses which do not cause direct protein damage, e.g. some genotoxic agents. These Stresses kill cells through activation of apoptosis, and Hsp72 increases cell survival by interfering with the apoptotic program. Recently it has been found that Hsp72 mediates suppression of a Stress-activated protein Kinase, JNK, an early component of Stress-induced apoptotic signalling pathway. This finding provides the basis for the anti-apoptotic activity of Hsp72. These observations can explain increased Stress sensitivity of aged cells in which compromised inducibility of Hsp72 leads to a loss of control of JNK activation by Stresses and subsequently to a higher rate of apoptotic death.
Phillip A Dennis - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylinositol ether lipid analogues that inhibit akt also independently activate the Stress Kinase p38α through mkk3 6 independent and dependent mechanisms
Journal of Biological Chemistry, 2007Co-Authors: Joell J Gills, Alan P. Kozikowski, Sianna S Castillo, Pavel A Petukhov, Chunyu Zhang, Melinda G Hollingshead, Regan M Memmott, Noel A Warfel, Jiahuai Han, Phillip A DennisAbstract:Abstract Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified Kinases. No Kinase was inhibited, but one isoform of p38, p38α, was uniformly activated 2-fold. Molecular modeling of p38α revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream Kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38α-/- cells, we showed that p38α is not required for PIA-induced apoptosis but is required for H2O2- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38α-/- cells increased apoptosis. These studies indicate that p38α is activated by PIAs through a novel mechanism and show that p38α activation contributes to PIA-induced cell death. Independent modulation of Akt and p38α could account for the profound cytotoxicity of PIAs.
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Importance of the Stress Kinase p38α in mediating the direct cytotoxic effects of the Thalidomide analogue, CPS49, in cancer cells and endothelial cells
Clinical Cancer Research, 2006Co-Authors: Noel A Warfel, Chunyu Zhang, Erin R. Lepper, William D. Figg, Phillip A DennisAbstract:Purpose: Thalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear. Experimental Design: To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of Kinases that control cellular survival in vitro and in vivo . Results: CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit Kinases that promote survival, such as Akt or extracellular signal-regulated Kinase, but rather rapidly activated the Stress Kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38α−/− cells or an inhibitor of p38, we show that the presence and activation of p38α is important for cytotoxicity in all cell types examined. Conclusions: Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.
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Importance of the Stress Kinase p38alpha in mediating the direct cytotoxic effects of the thalidomide analogue, CPS49, in cancer cells and endothelial cells.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2006Co-Authors: Noel A Warfel, Chunyu Zhang, Erin R. Lepper, William D. Figg, Phillip A DennisAbstract:Thalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear. To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of Kinases that control cellular survival in vitro and in vivo. CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit Kinases that promote survival, such as Akt or extracellular signal-regulated Kinase, but rather rapidly activated the Stress Kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38alpha-/- cells or an inhibitor of p38, we show that the presence and activation of p38alpha is important for cytotoxicity in all cell types examined. Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.
Angel Alonso - One of the best experts on this subject based on the ideXlab platform.
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Mechano-transduction in periodontal ligament cells identifies activated states of MAP-Kinases p42/44 and p38-Stress Kinase as a mechanism for MMP-13 expression
BMC Cell Biology, 2010Co-Authors: Nelli Ziegler, Angel Alonso, Thorsten Steinberg, Dale Woodnutt, Annette Kohl, Eva Müssig, Simon Schulz, Pascal TomakidiAbstract:Background Mechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated. Results In the present study we show by western blot (WB) analysis and/or indirect immunofluorescence (IIF) that mechanical strain modulates the amount of the matrix metalloproteinase MMP-13, and induces non-coherent modulation in the amount and activity of signal transducing molecules, such as FAK, MAP-Kinases p42/44, and p38 Stress Kinase, suggesting their mechanistic role in mechano-transduction. Increase in the amount of FAK occurs concomitant with increased levels of the focal contact integrin subunits β3 and β1, as indicated by WB or optionally by IIF. By employing specific inhibitors, we further identified p42/44 and p38 in their activated, i.e. phosphorylated state responsible for the expression of MMP-13. This finding may point to the obedience in the expression of this MMP as extracellular matrix (ECM) remodelling executioner from the activation state of mechano-transducing molecules. mRNA analysis by pathway-specific RT-profiler arrays revealed up- and/or down-regulation of genes assigning to MAP-Kinase signalling and cell cycle, ECM and integrins and growth factors. Up-regulated genes include for example focal contact integrin subunit α3, MMP-12, MAP-Kinases and associated Kinases, and the transcription factor c-fos, the latter as constituent of the AP1-complex addressing the MMP-13 promotor. Among others, genes down-regulated are those of COL-1 and COL-14, suggesting that strain-dependent mechano-transduction may transiently perturbate ECM homeostasis. Conclusions Strain-dependent mechano-/signal-transduction in PDL cells involves abundance and activity of FAK, MAP-Kinases p42/44, and p38 Stress Kinase in conjunction with the amount of MMP-13, and integrin subunits β1 and β3. Identifying the activated state of p42/44 and p38 as critical for MMP-13 expression may indicate the mechanistic contribution of mechano-transducing molecules on executioners of ECM homeostasis.
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mechano transduction in periodontal ligament cells identifies activated states of map Kinases p42 44 and p38 Stress Kinase as a mechanism for mmp 13 expression
BMC Cell Biology, 2010Co-Authors: Angel Alonso, Nelli Ziegler, Thorsten Steinberg, Dale Woodnutt, Annette Kohl, Eva Müssig, Simon Schulz, Pascal TomakidiAbstract:Mechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated. In the present study we show by western blot (WB) analysis and/or indirect immunofluorescence (IIF) that mechanical strain modulates the amount of the matrix metalloproteinase MMP-13, and induces non-coherent modulation in the amount and activity of signal transducing molecules, such as FAK, MAP-Kinases p42/44, and p38 Stress Kinase, suggesting their mechanistic role in mechano-transduction. Increase in the amount of FAK occurs concomitant with increased levels of the focal contact integrin subunits β3 and β1, as indicated by WB or optionally by IIF. By employing specific inhibitors, we further identified p42/44 and p38 in their activated, i.e. phosphorylated state responsible for the expression of MMP-13. This finding may point to the obedience in the expression of this MMP as extracellular matrix (ECM) remodelling executioner from the activation state of mechano-transducing molecules. mRNA analysis by pathway-specific RT-profiler arrays revealed up- and/or down-regulation of genes assigning to MAP-Kinase signalling and cell cycle, ECM and integrins and growth factors. Up-regulated genes include for example focal contact integrin subunit α3, MMP-12, MAP-Kinases and associated Kinases, and the transcription factor c-fos, the latter as constituent of the AP1-complex addressing the MMP-13 promotor. Among others, genes down-regulated are those of COL-1 and COL-14, suggesting that strain-dependent mechano-transduction may transiently perturbate ECM homeostasis. Strain-dependent mechano-/signal-transduction in PDL cells involves abundance and activity of FAK, MAP-Kinases p42/44, and p38 Stress Kinase in conjunction with the amount of MMP-13, and integrin subunits β1 and β3. Identifying the activated state of p42/44 and p38 as critical for MMP-13 expression may indicate the mechanistic contribution of mechano-transducing molecules on executioners of ECM homeostasis.
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Mechano-transduction in periodontal ligament cells identifies activated states of MAP-Kinases p42/44 and p38-Stress Kinase as a mechanism for MMP-13 expression
BMC cell biology, 2010Co-Authors: Nelli Ziegler, Angel Alonso, Thorsten Steinberg, Dale Woodnutt, Annette Kohl, Eva Müssig, Simon Schulz, Pascal TomakidiAbstract:Mechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated. In the present study we show by western blot (WB) analysis and/or indirect immunofluorescence (IIF) that mechanical strain modulates the amount of the matrix metalloproteinase MMP-13, and induces non-coherent modulation in the amount and activity of signal transducing molecules, such as FAK, MAP-Kinases p42/44, and p38 Stress Kinase, suggesting their mechanistic role in mechano-transduction. Increase in the amount of FAK occurs concomitant with increased levels of the focal contact integrin subunits β3 and β1, as indicated by WB or optionally by IIF. By employing specific inhibitors, we further identified p42/44 and p38 in their activated, i.e. phosphorylated state responsible for the expression of MMP-13. This finding may point to the obedience in the expression of this MMP as extracellular matrix (ECM) remodelling executioner from the activation state of mechano-transducing molecules. mRNA analysis by pathway-specific RT-profiler arrays revealed up- and/or down-regulation of genes assigning to MAP-Kinase signalling and cell cycle, ECM and integrins and growth factors. Up-regulated genes include for example focal contact integrin subunit α3, MMP-12, MAP-Kinases and associated Kinases, and the transcription factor c-fos, the latter as constituent of the AP1-complex addressing the MMP-13 promotor. Among others, genes down-regulated are those of COL-1 and COL-14, suggesting that strain-dependent mechano-transduction may transiently perturbate ECM homeostasis. Strain-dependent mechano-/signal-transduction in PDL cells involves abundance and activity of FAK, MAP-Kinases p42/44, and p38 Stress Kinase in conjunction with the amount of MMP-13, and integrin subunits β1 and β3. Identifying the activated state of p42/44 and p38 as critical for MMP-13 expression may indicate the mechanistic contribution of mechano-transducing molecules on executioners of ECM homeostasis.
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Multiple independent Kinase cascades are targeted by hyperosmotic Stress but only one activates Stress Kinase p38.
Experimental cell research, 2004Co-Authors: Xiaohong Mao, Hao Cheng, Ignacio G. Bravo, Angel AlonsoAbstract:Abstract In this report, we analyse the effects of osmotic shock on signal transduction in CHO cells. We demonstrate that at least three different Kinase cascades are switched on upon osmotic shock, namely PKA, AMPK, and MLTK. Whereas PKA from cells treated with forskolin activated Stress Kinase p38, PKA from cells treated with sorbitol did not activate p38, although the enzyme is activated in both cases as analysed in vitro using a specific peptide target. Further, osmolar shock activated AMPK but treatment of the cells with the AMPK activator 5-amino-4-imidazolecarboxamide (AICAr) did not result in p38 activation, strongly suggesting that AMPK is not involved in Stress Kinase activation. Transfection of CHO cells with dominant negative recombinants of MLTKα resulted in inhibition of sorbitol-mediated p38 activation, indicating that the mixed-lineage Kinase is involved in the activation of p38 by sorbitol. Finally, in CHO cells overexpressing wild-type MLTKα, no activation of AMPK of PKA could be demonstrated, indicating that the activated Kinase cascades are not involved in a cross-talk process.
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Stress Kinase p38 mediates EGFR transactivation by hyperosmolar concentrations of sorbitol
Journal of cellular physiology, 2002Co-Authors: Hao Cheng, Jürgen Kartenbeck, Kirsten Kabsch, Xiahong Mao, Margarita M. Marques, Angel AlonsoAbstract:Activation of the epidermal growth factor receptor (EGFR) has been shown to occur by ligand-dependent and ligand-independent mechanisms. Different molecular mechanisms have been found to be responsible for ligand-independent receptor transactivation. Here, we show that hyperosmolar concentrations of sorbitol activate the EGFR in human keratinocytes. Experiments using specific inhibitors of EGFR phosphorylation show that the increased amount of activated receptors is the result of a decreased rate of dephosphorylation. Furthermore, sorbitol treatment results in a strong activation of Stress Kinase p38. Treatment of the cells with SB203580, a known inhibitor of p38 α and β Kinases, results in impairment of receptor activation, indicating that the Stress Kinase is involved in receptor activation modulation. This is further reinforced by experiments showing that addition of Toxin B, known to be an inhibitor of the small Rho GTPases rac1, cdc42, and Rho A/B, to the cells results in a strong induction of EGFR activation. Our results point, therefore, to a mechanism by which osmotic shock activates EGFR through the small Rho GTPases-p38 Stress Kinase pathway. © 2002 Wiley-Liss, Inc.
Julia A. Yaglom - One of the best experts on this subject based on the ideXlab platform.
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Regulation of necrosis of H9c2 myogenic cells upon transient energy deprivation. Rapid deenergization of mitochondria precedes necrosis and is controlled by reactive oxygen species, Stress Kinase JNK, HSP72 and ARC.
The Journal of biological chemistry, 2003Co-Authors: Julia A. Yaglom, Vladimir L. Gabai, Daryoush Ekhterae, Michael Y. ShermanAbstract:Abstract Subjecting myogenic H9c2 cells to transient energy deprivation leads to a caspase-independent death with typical features of necrosis. Here we show that the rupture of cytoplasmic membrane, the terminal event in necrosis, is shortly preceded by rapid depolarization of mitochondrial membranes. The rapid deenergization of mitochondria critically depended upon prior generation of reactive oxygen species (ROS) during ATP depletion stage. Accordingly, expression of catalase prevented mitochondrial depolarization and averted subsequent necrosis. Interestingly, trifluoperazine, a compound that protects cells from ischemic insults, prevented necrosis of H9c2 cells through inhibition of ROS production. Other factors that regulated the mitochondrial membrane depolarization and subsequent loss of plasma membrane integrity include a Stress Kinase JNK activated at early steps of recovery from ATP depletion, as well as an apoptotic inhibitory protein ARC. Accordingly, inhibition of JNK or overexpression of ARC prevented mitochondrial depolarization and rescued H9c2 cells from necrosis. ROS and JNK affected mitochondrial deenergization and necrosis independently of each other since inhibition of ROS production did not prevent activation of JNK, whereas inhibition of JNK did not suppress ROS accumulation. Therefore, JNK activation and ROS production represent two independent pathways that control mitochondrial depolarization and subsequent necrosis of cells subjected to transient energy deprivation. Overexpression of ARC, although preventing mitochondrial depolarization, did not affect either JNK activation or production of ROS. The major heat shock protein Hsp72 inhibited JNK-related steps of necrotic pathway but did not affect ROS accumulation. Interestingly, mitochondrial depolarization and subsequent necrosis can be suppressed by an Hsp72 mutant Hsp72ΔEEVD, which lacks chaperone function but can efficiently suppress JNK activation. Thus, Hsp72 is directly implicated in a signaling pathway, which leads to necrotic death.
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Suppression of Stress Kinase JNK Is Involved in HSP72-mediated Protection of Myogenic Cells from Transient Energy Deprivation HSP72 ALLEVIATES THE Stress-INDUCED INHIBITION OF JNK DEPHOSPHORYLATION
The Journal of biological chemistry, 2000Co-Authors: Vladimir L. Gabai, Anatoli B. Meriin, Julia A. Yaglom, Jeanne Y. Wei, Dick D. Mosser, Michael Y. ShermanAbstract:Since protection of cells from Stress-induced apoptosis by the heat shock protein Hsp72 involves suppression of Stress Kinase JNK, we suggested that Hsp72-mediated JNK inhibition might also be critical for myocardial protection from ischemia/reperfusion. Transient energy deprivation of H9c2 myogenic cells, used as an in vitro model of myocardial ischemia, led to cell death that had morphological features of apoptosis and necrosis and was independent of caspases. Surprisingly, this unusual type of cell death was regulated by JNK and ERK Kinases. In fact, specific inhibition of JNK increased cell survival; specific inhibition of ERKs enhanced deleterious consequences of energy deprivation, whereas inhibition of p38 Kinase had no effect. Hsp72 suppressed activation of JNK and did not increase ERK activity, suggesting that inhibition of JNK is the important component of Hsp72-mediated protection. Upon transient energy deprivation, activation of JNK proceeds via two distinct pathways, stimulation of JNK phosphorylation by a protein Kinase SEK1 and inhibition of JNK dephosphorylation. Remarkably, in cells exposed to transient energy deprivation, Hsp72 enhanced the rate of JNK dephosphorylation but did not affect SEK1 activity. Therefore, it appears that Hsp72 specifically down-regulates JNK by accelerating its dephosphorylation, which reduces the susceptibility of cardiac cells to simulated ischemia/reperfusion.
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Role of Hsp70 in regulation of Stress-Kinase JNK: implications in apoptosis and aging.
FEBS letters, 1998Co-Authors: V L Gabai, Anatoli B. Meriin, Julia A. Yaglom, V Z Volloch, M Y ShermanAbstract:Cell protection from Stresses by the major heat shock protein Hsp72 was previously attributed to its ability to prevent aggregation and to accelerate refolding of damaged proteins. This repair function of Hsp72 may play an important role in cell survival after extremely harsh protein damaging treatments leading to necrotic cell death. On the other hand, protein repair function of Hsp72 cannot explain how it protects cells from Stresses which do not cause direct protein damage, e.g. some genotoxic agents. These Stresses kill cells through activation of apoptosis, and Hsp72 increases cell survival by interfering with the apoptotic program. Recently it has been found that Hsp72 mediates suppression of a Stress-activated protein Kinase, JNK, an early component of Stress-induced apoptotic signalling pathway. This finding provides the basis for the anti-apoptotic activity of Hsp72. These observations can explain increased Stress sensitivity of aged cells in which compromised inducibility of Hsp72 leads to a loss of control of JNK activation by Stresses and subsequently to a higher rate of apoptotic death.
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Role of Hsp70 in regulation of Stress‐Kinase JNK: implications in apoptosis and aging
FEBS Letters, 1998Co-Authors: Vladimir L. Gabai, Anatoli B. Meriin, Julia A. Yaglom, Vladimir Volloch, Michael Y. ShermanAbstract:Cell protection from Stresses by the major heat shock protein Hsp72 was previously attributed to its ability to prevent aggregation and to accelerate refolding of damaged proteins. This repair function of Hsp72 may play an important role in cell survival after extremely harsh protein damaging treatments leading to necrotic cell death. On the other hand, protein repair function of Hsp72 cannot explain how it protects cells from Stresses which do not cause direct protein damage, e.g. some genotoxic agents. These Stresses kill cells through activation of apoptosis, and Hsp72 increases cell survival by interfering with the apoptotic program. Recently it has been found that Hsp72 mediates suppression of a Stress-activated protein Kinase, JNK, an early component of Stress-induced apoptotic signalling pathway. This finding provides the basis for the anti-apoptotic activity of Hsp72. These observations can explain increased Stress sensitivity of aged cells in which compromised inducibility of Hsp72 leads to a loss of control of JNK activation by Stresses and subsequently to a higher rate of apoptotic death.
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Minireview Role of Hsp70 in regulation of Stress-Kinase JNK: implications in apoptosis and aging
1998Co-Authors: Vladimir L. Gabai, Anatoli B. Meriin, Julia A. Yaglom, Vladimir Volloch, Michael Y. ShermanAbstract:Cell protection from Stresses by the major heat shock protein Hsp72 was previously attributed to its ability to prevent aggregation and to accelerate refolding of damaged proteins. This repair function of Hsp72 may play an important role in cell survival after extremely harsh protein damaging treatments leading to necrotic cell death. On the other hand, protein repair function of Hsp72 cannot explain how it protects cells from Stresses which do not cause direct protein damage, e.g. some genotoxic agents. These Stresses kill cells through activation of apoptosis, and Hsp72 increases cell survival by interfering with the apoptotic program. Recently it has been found that Hsp72 mediates suppression of a Stress-activated protein Kinase, JNK, an early component of Stress-induced apoptotic signalling pathway. This finding provides the basis for the anti-apoptotic activity of Hsp72. These observations can explain increased Stress sensitivity of aged cells in which compromised inducibility of Hsp72 leads to a loss of control of JNK activation by Stresses and subsequently to a higher rate of apoptotic death. z 1998 Federation of European Biochemical Societies.