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BBA - Molecular Cell Research (v.1803, #3)
PTH inactivates the AKT survival pathway in the colonic cell line Caco-2 by Natalia Calvo; Ana Russo de Boland; Claudia Gentili (pp. 343-351).
In previous works, we found that PTH promotes the apoptosis of human Caco-2 intestinal cells, through the mitochondrial pathway. This study was conducted to investigate the modulation of different players implicated in the AKT survival pathway in PTH-induced intestinal cell apoptosis. We demonstrate, for the first time, that PTH modulates AKT phosphorylation in response to apoptosis via the serine/threonine phosphatase PP2A. PTH treatment induces an association of AKT with the catalytic subunit of PP2A and increases its phosphatase activity. PTH also promotes the translocation of PP2Ac from the cytosol to the mitochondria. Furthermore, our results suggest that PP2A plays a role in hormone-dependent Caco-2 cells viability and in the cleavage of caspase-3 and its substrate PARP. The cAMP pathway also contributes to PTH-mediated AKT dephosphorylation while PKC and p38 MAPK do not participate in this event. Finally, we show that PTH induces the dissociation between 14-3-3 and AKT, but the significance of this response remains unknown. In correlation with PTH-induced Bad dephosphorylation, the hormone also decreases the basal association of 14-3-3 and Bad. Overall, our data suggest that in Caco-2 cells, PP2A and the cAMP pathway act in concert to inactivate the AKT survival pathway in PTH-induced intestinal cell apoptosis.
Keywords: PTH; Caco-2 cell; Apoptosis; PP2A; AKT
PTH inactivates the AKT survival pathway in the colonic cell line Caco-2 by Natalia Calvo; Ana Russo de Boland; Claudia Gentili (pp. 343-351).
In previous works, we found that PTH promotes the apoptosis of human Caco-2 intestinal cells, through the mitochondrial pathway. This study was conducted to investigate the modulation of different players implicated in the AKT survival pathway in PTH-induced intestinal cell apoptosis. We demonstrate, for the first time, that PTH modulates AKT phosphorylation in response to apoptosis via the serine/threonine phosphatase PP2A. PTH treatment induces an association of AKT with the catalytic subunit of PP2A and increases its phosphatase activity. PTH also promotes the translocation of PP2Ac from the cytosol to the mitochondria. Furthermore, our results suggest that PP2A plays a role in hormone-dependent Caco-2 cells viability and in the cleavage of caspase-3 and its substrate PARP. The cAMP pathway also contributes to PTH-mediated AKT dephosphorylation while PKC and p38 MAPK do not participate in this event. Finally, we show that PTH induces the dissociation between 14-3-3 and AKT, but the significance of this response remains unknown. In correlation with PTH-induced Bad dephosphorylation, the hormone also decreases the basal association of 14-3-3 and Bad. Overall, our data suggest that in Caco-2 cells, PP2A and the cAMP pathway act in concert to inactivate the AKT survival pathway in PTH-induced intestinal cell apoptosis.
Keywords: PTH; Caco-2 cell; Apoptosis; PP2A; AKT
Calcium signaling of thyrocytes is modulated by TSH through calcium binding protein expression by Stephan Lorenz; Markus Eszlinger; Ralf Paschke; Gabriela Aust; Michael Weick; Fuhrer Dagmar Führer; Knut Krohn (pp. 352-360).
TSH is an important stimulus to maintain thyroid epithelial differentiation. Impairment of TSH signal transduction can cause thyroid pathologies such as hot nodules, goiter and hyperthyroidism. In a gene expression study in Fischer rat thyroid cells (FRTL-5) using cDNA microarrays we found a TSH-dependent regulation of several calcium binding proteins, S100A4, S100A6 and annexin A6. Expression of these genes in FRTL-5 and regulation by TSH was confirmed with LightCycler qPCR and Western blotting. The differential expression of S100A4 was confirmed for cultured primary human thyrocytes. Calcium-imaging experiments showed that prestimulation with TSH attenuates ATP-elicited P2Y-mediated calcium signaling. Experiments with thapsigargin, TSH and calcium-free perfusion excluded an involvement of other purinergic receptors or an involvement of SERCA regulation. Instead, we find a correlation between S100A4 expression and the effects of TSH on calcium signaling. Overexpression of S100A4 in FRTL-5 and shRNA-mediated knockdown of S100A4 in follicular thyroid cancer cells (FTC133) confirm the ability of S100A4 to attenuate calcium signals. Under repeated stimulations with ATP the calcium retention of these cells is also modulated by S100A4, suggesting a role of S100A4 as calcium buffering protein. As a biological consequence of S100A4 overexpression we detected reduced ATP-stimulated cFos induction. Taken together, the results suggest that S100A4 and other calcium binding proteins are part of a signaling network connecting TSH signaling to calcium-mediated events which play a role in thyroid physiology like H2O2 production or even thyroid cancer.
Keywords: Abbreviations; TSH; thyroid stimulating hormone, thyrotropin; hTSHR; human TSH receptor; CAM; Constitutively activating mutations; CaBP; Ca; 2+; binding proteinsS100A4 calcium binding protein; Fischer rat thyroid FRTL-5; Follicular thyroid cancer FTC133; Primary human thyrocyte; Microarray study; Calcium signaling; shRNA knockdown
Calcium signaling of thyrocytes is modulated by TSH through calcium binding protein expression by Stephan Lorenz; Markus Eszlinger; Ralf Paschke; Gabriela Aust; Michael Weick; Fuhrer Dagmar Führer; Knut Krohn (pp. 352-360).
TSH is an important stimulus to maintain thyroid epithelial differentiation. Impairment of TSH signal transduction can cause thyroid pathologies such as hot nodules, goiter and hyperthyroidism. In a gene expression study in Fischer rat thyroid cells (FRTL-5) using cDNA microarrays we found a TSH-dependent regulation of several calcium binding proteins, S100A4, S100A6 and annexin A6. Expression of these genes in FRTL-5 and regulation by TSH was confirmed with LightCycler qPCR and Western blotting. The differential expression of S100A4 was confirmed for cultured primary human thyrocytes. Calcium-imaging experiments showed that prestimulation with TSH attenuates ATP-elicited P2Y-mediated calcium signaling. Experiments with thapsigargin, TSH and calcium-free perfusion excluded an involvement of other purinergic receptors or an involvement of SERCA regulation. Instead, we find a correlation between S100A4 expression and the effects of TSH on calcium signaling. Overexpression of S100A4 in FRTL-5 and shRNA-mediated knockdown of S100A4 in follicular thyroid cancer cells (FTC133) confirm the ability of S100A4 to attenuate calcium signals. Under repeated stimulations with ATP the calcium retention of these cells is also modulated by S100A4, suggesting a role of S100A4 as calcium buffering protein. As a biological consequence of S100A4 overexpression we detected reduced ATP-stimulated cFos induction. Taken together, the results suggest that S100A4 and other calcium binding proteins are part of a signaling network connecting TSH signaling to calcium-mediated events which play a role in thyroid physiology like H2O2 production or even thyroid cancer.
Keywords: Abbreviations; TSH; thyroid stimulating hormone, thyrotropin; hTSHR; human TSH receptor; CAM; Constitutively activating mutations; CaBP; Ca; 2+; binding proteinsS100A4 calcium binding protein; Fischer rat thyroid FRTL-5; Follicular thyroid cancer FTC133; Primary human thyrocyte; Microarray study; Calcium signaling; shRNA knockdown
Microtubules govern stress granule mobility and dynamics by Elena S. Nadezhdina; Alexis J. Lomakin; Alexey A. Shpilman; Elena M. Chudinova; Pavel A. Ivanov (pp. 361-371).
Stress granules (SGs) are ribonucleoprotein (RNP)-containing assemblies that are formed in the cytoplasm in response to stress. Previously, we demonstrated that microtubule depolymerization inhibited SG formation. Here, we show that arsenate-induced SGs move throughout the cytoplasm in a microtubule-dependent manner, and microtubules are required for SG disassembly, but not for SG persistence. Analysis of SG movement revealed that SGs exhibited obstructed diffusion on an average, though sometimes SGs demonstrated rapid displacements. Microtubule depolymerization did not influence preformed SG number and size, but significantly reduced the average velocity of SG movement, the frequency of quick movement events, and the apparent diffusion coefficient of SGs. Actin filament disruption had no effect on the SG motility. In cycloheximide-treated cells SGs dissociated into constituent parts that then dissolved within the cytoplasm. Microtubule depolymerization inhibited cycloheximide-induced SG disassembly. However, microtubule depolymerization did not influence the dynamics of poly(A)-binding protein (PABP) in SGs, according to FRAP results. We suggest that the increase of SG size is facilitated by the transport of smaller SGs along microtubules with subsequent fusion of them. At least some protein components of SGs can exchange with the cytoplasmic pool independently of microtubules.
Keywords: RNP; Stress granules; Arsenate; Microtubule; FRAP; PABP
Microtubules govern stress granule mobility and dynamics by Elena S. Nadezhdina; Alexis J. Lomakin; Alexey A. Shpilman; Elena M. Chudinova; Pavel A. Ivanov (pp. 361-371).
Stress granules (SGs) are ribonucleoprotein (RNP)-containing assemblies that are formed in the cytoplasm in response to stress. Previously, we demonstrated that microtubule depolymerization inhibited SG formation. Here, we show that arsenate-induced SGs move throughout the cytoplasm in a microtubule-dependent manner, and microtubules are required for SG disassembly, but not for SG persistence. Analysis of SG movement revealed that SGs exhibited obstructed diffusion on an average, though sometimes SGs demonstrated rapid displacements. Microtubule depolymerization did not influence preformed SG number and size, but significantly reduced the average velocity of SG movement, the frequency of quick movement events, and the apparent diffusion coefficient of SGs. Actin filament disruption had no effect on the SG motility. In cycloheximide-treated cells SGs dissociated into constituent parts that then dissolved within the cytoplasm. Microtubule depolymerization inhibited cycloheximide-induced SG disassembly. However, microtubule depolymerization did not influence the dynamics of poly(A)-binding protein (PABP) in SGs, according to FRAP results. We suggest that the increase of SG size is facilitated by the transport of smaller SGs along microtubules with subsequent fusion of them. At least some protein components of SGs can exchange with the cytoplasmic pool independently of microtubules.
Keywords: RNP; Stress granules; Arsenate; Microtubule; FRAP; PABP
Endothelial nitric oxide synthase is essential for nitric oxide generation, L-type Ca2+ channel activation and survival in RBL-2H3 mast cells by Yoshihiro Suzuki; Toshio Inoue; Chisei Ra (pp. 372-385).
Recent pharmacological and molecular genetic approaches have revealed the existence of functional L-type Ca2+ channels (LTCCs) in a variety of hematopoietic cells. We previously reported that Cav1.2 LTCCs are expressed on mast cell surfaces, activated by the high-affinity IgE receptor (FcɛRI) engagement and protect mast cells against activation-induced cell death (AICD). We also demonstrated that FcɛRI engagement evokes nitric oxide (NO) generation in a phosphatidylinositol-3-kinase- and NO synthase (NOS)-dependent manner, which is also required for mast cell survival. Here we demonstrate that this endogenous NO mediates Cav1.2 LTCC activation. FcɛRI engagement but not thapsigargin, a potent Ca2+ release-activated Ca2+ (CRAC) channel agonist, induced Ca2+ influx via NOS-dependent NO generation. RT-PCR analyses revealed predominant expression of eNOS in mast cells. Subsequent experiments involving siRNA-mediated gene silencing of eNOS or Cav1.2 LTCC revealed that eNOS was essential for NOS-dependent NO generation and Cav1.2 LTCC activation but not CRAC channel activation. Similar to Cav1.2 LTCCs, eNOS prevented the dissipation of the mitochondrial membrane potential and mitochondrial integrity collapse, thereby protecting mast cells against AICD. Taken together, the present findings demonstrate the key roles of the eNOS-NO-LTCC axis in mast cell survival after FcɛRI engagement.
Keywords: Endothelial NOS; Nitric oxide; Ca; 2+; Mitochondria; Survival; Mast cell
Endothelial nitric oxide synthase is essential for nitric oxide generation, L-type Ca2+ channel activation and survival in RBL-2H3 mast cells by Yoshihiro Suzuki; Toshio Inoue; Chisei Ra (pp. 372-385).
Recent pharmacological and molecular genetic approaches have revealed the existence of functional L-type Ca2+ channels (LTCCs) in a variety of hematopoietic cells. We previously reported that Cav1.2 LTCCs are expressed on mast cell surfaces, activated by the high-affinity IgE receptor (FcɛRI) engagement and protect mast cells against activation-induced cell death (AICD). We also demonstrated that FcɛRI engagement evokes nitric oxide (NO) generation in a phosphatidylinositol-3-kinase- and NO synthase (NOS)-dependent manner, which is also required for mast cell survival. Here we demonstrate that this endogenous NO mediates Cav1.2 LTCC activation. FcɛRI engagement but not thapsigargin, a potent Ca2+ release-activated Ca2+ (CRAC) channel agonist, induced Ca2+ influx via NOS-dependent NO generation. RT-PCR analyses revealed predominant expression of eNOS in mast cells. Subsequent experiments involving siRNA-mediated gene silencing of eNOS or Cav1.2 LTCC revealed that eNOS was essential for NOS-dependent NO generation and Cav1.2 LTCC activation but not CRAC channel activation. Similar to Cav1.2 LTCCs, eNOS prevented the dissipation of the mitochondrial membrane potential and mitochondrial integrity collapse, thereby protecting mast cells against AICD. Taken together, the present findings demonstrate the key roles of the eNOS-NO-LTCC axis in mast cell survival after FcɛRI engagement.
Keywords: Endothelial NOS; Nitric oxide; Ca; 2+; Mitochondria; Survival; Mast cell
Chk2 splice variants express a dominant-negative effect on the wild-type Chk2 kinase activity by Elisabet Ognedal Berge; Vidar Staalesen; Anne Hege Straume; Johan Richard Lillehaug; Lonning Per Eystein Lønning (pp. 386-395).
While the majority of RNA transcripts from protein-encoding genes in the human genome are subject to physiological splicing, pathological splicing is increasingly reported in cancer tissue. Previously, we identified >90 different splice variants of Chk2, a gene encoding a serine/threonine kinase propagating the DNA damage signal by phosphorylating and activating several downstream substrates like p53, Cdc25A, and Cdc25C involved in cell cycle arrest and apoptosis. While alternative splice forms of other genes have been reported to exert a dominant-negative effect on the wild-type molecules, the function of Chk2 splice protein variants is still unclear. Here we evaluated the function of four Chk2 splice proteins for which mRNA splice variants were identified in human breast carcinomas. These splice variants were stably expressed as nuclear proteins. Two splice forms (Chk2Δ4 and Chk2del(2-3)) expressed kinase activity while variants Chk2Δ11 and Chk2isoI were essentially kinase inactive. Independent of intrinsic kinase activity, each splice variant impaired wild-type Chk2 activity through heterodimerization. Based on our findings, we suggest alternative splicing as a possible novel mechanism for repression of the Chk2 wild-type function .
Keywords: Abbreviations; Chk2; checkpoint kinase 2; Chk2wt; Chk2 wild-type; FHA domain; fork-head associated domain; KD; kinase dead; LFS; Li–Fraumeni syndrome; RF; reading frame; SCD; SQ/TQ cluster domain; TP53; p53 tumor suppressor geneChk2; Splicing; Breast cancer
Chk2 splice variants express a dominant-negative effect on the wild-type Chk2 kinase activity by Elisabet Ognedal Berge; Vidar Staalesen; Anne Hege Straume; Johan Richard Lillehaug; Lonning Per Eystein Lønning (pp. 386-395).
While the majority of RNA transcripts from protein-encoding genes in the human genome are subject to physiological splicing, pathological splicing is increasingly reported in cancer tissue. Previously, we identified >90 different splice variants of Chk2, a gene encoding a serine/threonine kinase propagating the DNA damage signal by phosphorylating and activating several downstream substrates like p53, Cdc25A, and Cdc25C involved in cell cycle arrest and apoptosis. While alternative splice forms of other genes have been reported to exert a dominant-negative effect on the wild-type molecules, the function of Chk2 splice protein variants is still unclear. Here we evaluated the function of four Chk2 splice proteins for which mRNA splice variants were identified in human breast carcinomas. These splice variants were stably expressed as nuclear proteins. Two splice forms (Chk2Δ4 and Chk2del(2-3)) expressed kinase activity while variants Chk2Δ11 and Chk2isoI were essentially kinase inactive. Independent of intrinsic kinase activity, each splice variant impaired wild-type Chk2 activity through heterodimerization. Based on our findings, we suggest alternative splicing as a possible novel mechanism for repression of the Chk2 wild-type function .
Keywords: Abbreviations; Chk2; checkpoint kinase 2; Chk2wt; Chk2 wild-type; FHA domain; fork-head associated domain; KD; kinase dead; LFS; Li–Fraumeni syndrome; RF; reading frame; SCD; SQ/TQ cluster domain; TP53; p53 tumor suppressor geneChk2; Splicing; Breast cancer
Single ovalbumin molecules exploring nucleoplasm and nucleoli of living cell nuclei by Jasmin Speil; Ulrich Kubitscheck (pp. 396-404).
The nucleus is the center of direction and coordination of the cell's metabolic and reproductive activities and contains numerous functionally specialized domains. These subnuclear structures are not delimited by membranes like cytoplasmic organelles and their function is only poorly understood. Here, we studied the most prominent nuclear domains, nucleoli and the remaining nucleoplasm. We used fluorescently labeled ovalbumin-ATTO647N, an inert protein, to examine their physical properties. This inert tracer was microinjected into the cytoplasm of HeLa cells, and after diffusion into the nucleus the tracer distribution and mobility in the two nuclear compartments was examined. Like many macromolecular probes ovalbumin was significantly less abundant in nucleoli compared to the nucleoplasm. High-speed fluorescence microscopy allowed visualizing and analyzing single tracer molecule trajectories within nucleoli and nucleoplasm. In accordance with previous studies we found that the viscosity of the nucleus is sevenfold higher than that of aqueous buffer. Notably, nucleoplasm and nucleoli did not significantly differ in viscosity, however, the fraction of slow or trapped molecules was higher in the nucleoplasm than in nucleoli (6% versus 0.2%). Surprisingly, even a completely inert molecule like ovalbumin showed at times short-lived binding events with a decay time of 8 ms in the nucleoplasm and even shorter–6.3 ms–within the nucleoli.
Keywords: Fluorescence microscopy; Single molecule microscopy; Nucleus; Nucleolus; Molecular mobility; Diffusion
Single ovalbumin molecules exploring nucleoplasm and nucleoli of living cell nuclei by Jasmin Speil; Ulrich Kubitscheck (pp. 396-404).
The nucleus is the center of direction and coordination of the cell's metabolic and reproductive activities and contains numerous functionally specialized domains. These subnuclear structures are not delimited by membranes like cytoplasmic organelles and their function is only poorly understood. Here, we studied the most prominent nuclear domains, nucleoli and the remaining nucleoplasm. We used fluorescently labeled ovalbumin-ATTO647N, an inert protein, to examine their physical properties. This inert tracer was microinjected into the cytoplasm of HeLa cells, and after diffusion into the nucleus the tracer distribution and mobility in the two nuclear compartments was examined. Like many macromolecular probes ovalbumin was significantly less abundant in nucleoli compared to the nucleoplasm. High-speed fluorescence microscopy allowed visualizing and analyzing single tracer molecule trajectories within nucleoli and nucleoplasm. In accordance with previous studies we found that the viscosity of the nucleus is sevenfold higher than that of aqueous buffer. Notably, nucleoplasm and nucleoli did not significantly differ in viscosity, however, the fraction of slow or trapped molecules was higher in the nucleoplasm than in nucleoli (6% versus 0.2%). Surprisingly, even a completely inert molecule like ovalbumin showed at times short-lived binding events with a decay time of 8 ms in the nucleoplasm and even shorter–6.3 ms–within the nucleoli.
Keywords: Fluorescence microscopy; Single molecule microscopy; Nucleus; Nucleolus; Molecular mobility; Diffusion
Infection with AV-SUR2A protects H9C2 cells against metabolic stress: A mechanism of SUR2A-mediated cytoprotection independent from the KATP channel activity by Qingyou Du; Sofija Jovanović; Andriy Sukhodub; Aleksandar Jovanović (pp. 405-415).
Transgenic mice overexpressing SUR2A, a subunit of ATP-sensitive K+ (KATP) channels, acquire resistance to myocardial ischaemia. However, the mechanism of SUR2A-mediated cytoprotection is yet to be fully understood. Adenoviral SUR2A construct (AV-SUR2A) increased SUR2A expression, number of KATP channels and subsarcolemmal ATP in glycolysis-sensitive manner in H9C2 cells. It also increased K+ current in response to chemical hypoxia, partially preserved subsarcolemmal ATP and increased cell survival. Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K+ conductance, abolished the effect of SUR2A on K+ current, did not affect SUR2A-induced increase in subsarcolemmal ATP and partially inhibited SUR2A-mediated cytoprotection. Infection with 193gly-M-LDH, an inactive mutant of muscle lactate dehydrogenase, abolished the effect of SUR2A on K+ current, subsarcolemmal ATP and cell survival; the effect of 193gly-M-LDH on cell survival was significantly more pronounced than those of Kir6.2AFA. We conclude that AV-SUR2A increases resistance to metabolic stress in H9C2 cells by increasing the number of sarcolemmal KATP channels and subsarcolemmal ATP.
Keywords: SUR2A; ATP; K; ATP; channel; Cardioprotection; Ischaemia; Heart
Infection with AV-SUR2A protects H9C2 cells against metabolic stress: A mechanism of SUR2A-mediated cytoprotection independent from the KATP channel activity by Qingyou Du; Sofija Jovanović; Andriy Sukhodub; Aleksandar Jovanović (pp. 405-415).
Transgenic mice overexpressing SUR2A, a subunit of ATP-sensitive K+ (KATP) channels, acquire resistance to myocardial ischaemia. However, the mechanism of SUR2A-mediated cytoprotection is yet to be fully understood. Adenoviral SUR2A construct (AV-SUR2A) increased SUR2A expression, number of KATP channels and subsarcolemmal ATP in glycolysis-sensitive manner in H9C2 cells. It also increased K+ current in response to chemical hypoxia, partially preserved subsarcolemmal ATP and increased cell survival. Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K+ conductance, abolished the effect of SUR2A on K+ current, did not affect SUR2A-induced increase in subsarcolemmal ATP and partially inhibited SUR2A-mediated cytoprotection. Infection with 193gly-M-LDH, an inactive mutant of muscle lactate dehydrogenase, abolished the effect of SUR2A on K+ current, subsarcolemmal ATP and cell survival; the effect of 193gly-M-LDH on cell survival was significantly more pronounced than those of Kir6.2AFA. We conclude that AV-SUR2A increases resistance to metabolic stress in H9C2 cells by increasing the number of sarcolemmal KATP channels and subsarcolemmal ATP.
Keywords: SUR2A; ATP; K; ATP; channel; Cardioprotection; Ischaemia; Heart
Proteasomal inhibition upregulates the endogenous MAPK deactivator MKP-1 in human airway smooth muscle: Mechanism of action and effect on cytokine secretion by John P. Moutzouris; Wenchi Che; Emma E. Ramsay; Melanie Manetsch; Hatem Alkhouri; Anna M. Bjorkman; Friederike Schuster; Qi Ge; Alaina J. Ammit (pp. 416-423).
Asthma is a chronic inflammatory condition. Inhibition of the ubiquitin–proteasome system offers promise as a anti-inflammatory strategy, being responsible for the degradation of key proteins involved in crucial cellular functions, including gene expression in inflammation (e.g. inhibitory IκB-α and the endogenous MAPK deactivator — MKP-1). As MKP-1 inhibits MAPK-mediated pro-remodeling functions in human airway smooth muscle (ASM; a pivotal immunomodulatory cell in asthma) in this study we investigate the effect of the proteasome inhibitor MG-132 on MKP-1 and evaluate the anti-inflammatory effect of MG-132 on cytokine secretion from ASM cells. Examining the time-course of induction of MKP-1 mRNA and protein by MG-132 (10μM) we show that MKP-1 mRNA was first detected at 30min, increased to significant levels by 4h, resulting in a 12.6±1.5-fold increase in MKP-1 mRNA expression by 24h ( P<0.05). MKP-1 protein levels corroborate the mRNA results. Investigating the effect of MG-132 on secretion of the cytokine IL-6 we show that while short-term pretreatment with MG-132 (30min) partially reduced TNFα-induced IL-6 via inhibition of IκB-α degradation and the NF-κB pathway, longer-term proteasome inhibition (up to 24h) robustly upregulated MKP-1 and was temporally correlated with repression of p38-mediated IL-6 secretion from ASM cells. Moreover, utilizing a cytokine array we show that MG-132 represses the secretion of multiple cytokines implicated in asthma. Taken together, our results demonstrate that MG-132 upregulates MKP-1 and represses cytokine secretion from ASM and highlight the potential of the proteasome as a therapeutic target in asthma.
Keywords: MG-132; MKP-1; MAPK; IκB-α; NF-κB
Proteasomal inhibition upregulates the endogenous MAPK deactivator MKP-1 in human airway smooth muscle: Mechanism of action and effect on cytokine secretion by John P. Moutzouris; Wenchi Che; Emma E. Ramsay; Melanie Manetsch; Hatem Alkhouri; Anna M. Bjorkman; Friederike Schuster; Qi Ge; Alaina J. Ammit (pp. 416-423).
Asthma is a chronic inflammatory condition. Inhibition of the ubiquitin–proteasome system offers promise as a anti-inflammatory strategy, being responsible for the degradation of key proteins involved in crucial cellular functions, including gene expression in inflammation (e.g. inhibitory IκB-α and the endogenous MAPK deactivator — MKP-1). As MKP-1 inhibits MAPK-mediated pro-remodeling functions in human airway smooth muscle (ASM; a pivotal immunomodulatory cell in asthma) in this study we investigate the effect of the proteasome inhibitor MG-132 on MKP-1 and evaluate the anti-inflammatory effect of MG-132 on cytokine secretion from ASM cells. Examining the time-course of induction of MKP-1 mRNA and protein by MG-132 (10μM) we show that MKP-1 mRNA was first detected at 30min, increased to significant levels by 4h, resulting in a 12.6±1.5-fold increase in MKP-1 mRNA expression by 24h ( P<0.05). MKP-1 protein levels corroborate the mRNA results. Investigating the effect of MG-132 on secretion of the cytokine IL-6 we show that while short-term pretreatment with MG-132 (30min) partially reduced TNFα-induced IL-6 via inhibition of IκB-α degradation and the NF-κB pathway, longer-term proteasome inhibition (up to 24h) robustly upregulated MKP-1 and was temporally correlated with repression of p38-mediated IL-6 secretion from ASM cells. Moreover, utilizing a cytokine array we show that MG-132 represses the secretion of multiple cytokines implicated in asthma. Taken together, our results demonstrate that MG-132 upregulates MKP-1 and represses cytokine secretion from ASM and highlight the potential of the proteasome as a therapeutic target in asthma.
Keywords: MG-132; MKP-1; MAPK; IκB-α; NF-κB
Clearance of RhodopsinP23H aggregates requires the ERAD effector VCP by Ana Griciuc; Liviu Aron; Giovanni Piccoli; Marius Ueffing (pp. 424-434).
Dominant mutations in the visual pigment Rhodopsin (Rh) cause retinitis pigmentosa (RP) characterized by progressive blindness and retinal degeneration. The most common Rh mutation, RhP23H forms aggregates in the endoplasmic reticulum (ER) and impairs the proteasome; however, the mechanisms linking Rh aggregate formation to proteasome dysfunction and photoreceptor cell loss remain unclear. Using mammalian cell cultures, we provide the first evidence that misfolded RhP23H is a substrate of the ERAD effector VCP, an ATP-dependent chaperone that extracts misfolded proteins from the ER and escorts them for proteasomal degradation. VCP co-localizes with misfolded RhP23H in retinal cells and requires functional N-terminal and D1 ATPase domains to form a complex with RhP23H aggregates. Furthermore, VCP uses its D2 ATPase activity to promote RhP23H aggregate retrotranslocation and proteasomal delivery. Our results raise the possibility that modulation of VCP and ERAD activity might have potential therapeutic significance for RP.
Keywords: ATPase; ERAD; Proteasome; Rhodopsin; Retinitis pigmentosa; VCP
Clearance of RhodopsinP23H aggregates requires the ERAD effector VCP by Ana Griciuc; Liviu Aron; Giovanni Piccoli; Marius Ueffing (pp. 424-434).
Dominant mutations in the visual pigment Rhodopsin (Rh) cause retinitis pigmentosa (RP) characterized by progressive blindness and retinal degeneration. The most common Rh mutation, RhP23H forms aggregates in the endoplasmic reticulum (ER) and impairs the proteasome; however, the mechanisms linking Rh aggregate formation to proteasome dysfunction and photoreceptor cell loss remain unclear. Using mammalian cell cultures, we provide the first evidence that misfolded RhP23H is a substrate of the ERAD effector VCP, an ATP-dependent chaperone that extracts misfolded proteins from the ER and escorts them for proteasomal degradation. VCP co-localizes with misfolded RhP23H in retinal cells and requires functional N-terminal and D1 ATPase domains to form a complex with RhP23H aggregates. Furthermore, VCP uses its D2 ATPase activity to promote RhP23H aggregate retrotranslocation and proteasomal delivery. Our results raise the possibility that modulation of VCP and ERAD activity might have potential therapeutic significance for RP.
Keywords: ATPase; ERAD; Proteasome; Rhodopsin; Retinitis pigmentosa; VCP