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Rubinelli L, Manzo OL, Sungho J, Del Gaudio I, Bareja R, Marino A, Palikhe S, Di Mauro V, Bucci M, Falcone DJ, Elemento O, Ersoy B, Diano S, Sasset L, Di Lorenzo A. Suppression of endothelial ceramide de novo biosynthesis by Nogo-B contributes to cardiometabolic diseases. Nat Commun 2025; 16:1968. [PMID: 40000621 PMCID: PMC11862206 DOI: 10.1038/s41467-025-56869-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Accepted: 02/04/2025] [Indexed: 02/27/2025] Open
Abstract
Accrual of ceramides, membrane and bioactive sphingolipids, has been implicated in endothelial dysfunction preceding cardiometabolic diseases. Yet, direct in vivo evidence, underlying mechanisms, and pathological implications are lacking. Here we show that suppression of ceramides and sphingosine-1-phosphate (S1P), a product of ceramide degradation, are causally linked to endothelial dysfunction and activation, contributing to vascular and metabolic disorders in high fat diet fed (HFD) male mice. Mechanistically, the upregulation of Nogo-B and ORMDL proteins suppress ceramide de novo biosynthesis in endothelial cells (EC) of HFD mice, resulting in vascular and metabolic dysfunctions. Systemic and endothelial specific deletion of Nogo-B restore sphingolipid signaling and functions, lowers hypertension, and hepatic glucose production in HFD. Our results demonstrate in vivo that ceramide and S1P suppression rather than accrual contributes to endothelial dysfunction and cardiometabolic diseases in HFD mice. Our study also sets a framework for the development of therapeutic strategies to treat these conditions.
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Affiliation(s)
- Luisa Rubinelli
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
- Cardiovascular Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Onorina Laura Manzo
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
- Cardiovascular Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Jin Sungho
- Institute of Human Nutrition, Columbia University Irving Medical Center, New York, NY, USA
| | - Ilaria Del Gaudio
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
- Cardiovascular Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Rohan Bareja
- The Caryl and Israel Englander Institute for Precision Medicine, Weill Cornell Medicine and New York-Presbyterian Hospital, New York, NY, USA
| | - Alice Marino
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
- Cardiovascular Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Sailesh Palikhe
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
- Cardiovascular Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Vittoria Di Mauro
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
- Cardiovascular Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Mariarosaria Bucci
- Department of Pharmacy, School of Medicine, University of Naples Federico II, Naples, Italy
| | - Domenick J Falcone
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Olivier Elemento
- The Caryl and Israel Englander Institute for Precision Medicine, Weill Cornell Medicine and New York-Presbyterian Hospital, New York, NY, USA
| | - Baran Ersoy
- Joan & Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Sabrina Diano
- Institute of Human Nutrition, Columbia University Irving Medical Center, New York, NY, USA
| | - Linda Sasset
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA
- Cardiovascular Research Institute, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Annarita Di Lorenzo
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA.
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Restoration of ceramide de novo synthesis by the synthetic retinoid ST1926 as it induces adult T-cell leukemia cell death. Biosci Rep 2021; 40:226649. [PMID: 33048123 PMCID: PMC7593536 DOI: 10.1042/bsr20200050] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2020] [Revised: 09/21/2020] [Accepted: 09/30/2020] [Indexed: 01/15/2023] Open
Abstract
Ceramide (Cer) is a bioactive cellular lipid with compartmentalized and tightly regulated levels. Distinct metabolic pathways lead to the generation of Cer species with distinguishable roles in oncogenesis. Deregulation of Cer pathways has emerged as an important mechanism for acquired chemotherapeutic resistance. Adult T-cell leukemia (ATL) cells are defective in Cer synthesis. ATL is an aggressive neoplasm that develops following infection with human T-cell lymphotropic virus-1 (HTLV-1) where the viral oncogene Tax contributes to the pathogenesis of the disease. ATL cells, resistant to all-trans-retinoic acid, are sensitive to pharmacologically achievable concentrations of the synthetic retinoid ST1926. We studied the effects of ST1926 on Cer pathways in ATL cells. ST1926 treatment resulted in early Tax oncoprotein degradation in HTLV-1-treated cells. ST1926 induced cell death and a dose- and time-dependent accumulation of Cer in malignant T cells. The kinetics and degree of Cer production showed an early response upon ST1926 treatment. ST1926 enhanced de novo Cer synthesis via activation of ceramide synthase CerS(s) without inhibiting dihydroceramide desaturase, thereby accumulating Cer rather than the less bioactive dihydroceramide. Using labeling experiments with the unnatural 17-carbon sphinganine and measuring the generated Cer species, we showed that ST1926 preferentially induces the activities of a distinct set of CerS(s). We detected a delay in cell death response and interruption of Cer generation in response to ST1926 in Molt-4 cells overexpressing Bcl-2. These results highlight the potential role of ST1926 in inducing Cer levels, thus lowering the threshold for cell death in ATL cells.
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Prymas K, Świątkowska A, Traczyk G, Ziemlińska E, Dziewulska A, Ciesielska A, Kwiatkowska K. Sphingomyelin synthase activity affects TRIF-dependent signaling of Toll-like receptor 4 in cells stimulated with lipopolysaccharide. Biochim Biophys Acta Mol Cell Biol Lipids 2020; 1865:158549. [DOI: 10.1016/j.bbalip.2019.158549] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Revised: 09/10/2019] [Accepted: 09/25/2019] [Indexed: 01/08/2023]
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Hage-Sleiman R, Hamze AB, El-Hed AF, Attieh R, Kozhaya L, Kabbani S, Dbaibo G. Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells. Immunol Res 2017; 64:869-86. [PMID: 26798039 DOI: 10.1007/s12026-016-8787-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase's translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation-where PKCθ plays a critical role.
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Affiliation(s)
- Rouba Hage-Sleiman
- Department of Biology, Faculty of Sciences, Lebanese University, Hadath, Lebanon
| | - Asmaa B Hamze
- Department of Biomedical Science, Faculty of Health Sciences, Global University, Batrakiyye, Beirut, Lebanon
| | - Aimée F El-Hed
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon
| | - Randa Attieh
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon
| | - Lina Kozhaya
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon
| | - Sarah Kabbani
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon
| | - Ghassan Dbaibo
- Department of Pediatrics and Adolescent Medicine, Center for Infectious Diseases Research, Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, PO Box 11-0236 Riad El Solh, Beirut, Lebanon.
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C6-ceramide nanoliposome suppresses tumor metastasis by eliciting PI3K and PKCζ tumor-suppressive activities and regulating integrin affinity modulation. Sci Rep 2015; 5:9275. [PMID: 25792190 PMCID: PMC4366857 DOI: 10.1038/srep09275] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2014] [Accepted: 02/02/2015] [Indexed: 01/07/2023] Open
Abstract
Nanoliposomal formulation of C6-ceramide, a proapoptotic sphingolipid metabolite, presents an effective way to treat malignant tumor. Here, we provide evidence that acute treatment (30 min) of melanoma and breast cancer cells with nanoliposomal C6-ceramide (NaL-C6) may suppress cell migration without inducing cell death. By employing a novel flow migration assay, we demonstrated that NaL-C6 decreased tumor extravasation under shear conditions. Compared with ghost nanoliposome, NaL-C6 triggered phosphorylation of PI3K and PKCζ and dephosphorylation of PKCα. Concomitantly, activated PKCζ translocated into cell membrane. siRNA knockdown or pharmacological inhibition of PKCζ or PI3K rescued NaL-C6-mediated suppression of tumor migration. By inducing dephosphorylation of paxillin, PKCζ was responsible for NaL-C6-mediated stress fiber depolymerization and focal adhesion disassembly in the metastatic tumor cells. PKCζ and PI3K regulated cell shear-resistant adhesion in a way that required integrin αvβ3 affinity modulation. In conclusion, we identified a novel role of acute nanoliposomal ceramide treatment in reducing integrin affinity and inhibiting melanoma metastasis by conferring PI3K and PKCζ tumor-suppressive activities.
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Zanabria R, Tellez AM, Griffiths M, Corredig M. Milk fat globule membrane isolate induces apoptosis in HT-29 human colon cancer cells. Food Funct 2013; 4:222-30. [DOI: 10.1039/c2fo30189j] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Dakroub Z, Kreydiyyeh SI. Sphingosine-1-phosphate is a mediator of TNF-α action on the Na+/K+ ATPase in HepG2 cells. J Cell Biochem 2012; 113:2077-85. [PMID: 22271589 DOI: 10.1002/jcb.24079] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
We showed previously that TNF-α down-regulates the Na+/K+ ATPase in HepG2 cells. This work was undertaken to study the role of ceramide and its metabolites in TNF-α action. Treating HepG2 cells with the cytokine in presence of an inhibitor of sphingomyelinase, abrogated the effect of TNF-α on the ATPase. To confirm the involvement of ceramide or its metabolites, cells were incubated with exogenous ceramide. Ceramide reduced time-dependently the activity of the ATPase and its effect disappeared in presence of CAY 10466 or SHKI, respective inhibitors of ceramidase and spingosine kinase, suggesting that ceramide acts via sphingosine or sphingosine-1-phosphate (S1P). However, HepG2 cells treated with exogenous sphingosine showed a higher Na+/K+ ATPase activity inferring that S1P is the one responsible for the down-regulatory effect of TNF-α and ceramide. This hypothesis was confirmed by the observed inhibitory effect of exogenous S1P on the pump, which was maintained when JNK and NF-κB were inhibited separately or simultaneously. The concurrent, but not individual inhibition of the kinase and transcription factor in the absence of S1P imitated the effect of S1P. It was concluded that S1P down-regulates the ATPase by inhibiting both JNK and NF-κB. This conclusion was supported by the observed decrease in the phosphorylation of c-jun and the enhanced protein expression of IκB and lower NK-KB activity.
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Affiliation(s)
- Zeina Dakroub
- Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon
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Curry MC, Luk NA, Kenny PA, Roberts-Thomson SJ, Monteith GR. Distinct regulation of cytoplasmic calcium signals and cell death pathways by different plasma membrane calcium ATPase isoforms in MDA-MB-231 breast cancer cells. J Biol Chem 2012; 287:28598-608. [PMID: 22733819 DOI: 10.1074/jbc.m112.364737] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.
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Affiliation(s)
- Merril C Curry
- School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072, Australia
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Zhou CH, Xiang M, He SY, Qian ZY. Protein kinase C pathway is involved in the inhibition by crocetin of vascular smooth muscle cells proliferation. Phytother Res 2010; 24:1680-6. [DOI: 10.1002/ptr.3194] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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The inhibition of lipopolysaccharide-induced tumor necrosis factor-α and nitric oxide production by Clostridium perfringens α-toxin and its relation to α-toxin-induced intracellular ceramide generation. Int J Med Microbiol 2009; 299:554-62. [DOI: 10.1016/j.ijmm.2009.04.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2009] [Revised: 03/04/2009] [Accepted: 04/19/2009] [Indexed: 11/18/2022] Open
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High vapor pressure perfluorocarbons cause vesicle fusion and changes in membrane packing. Biophys J 2008; 95:4737-47. [PMID: 18689464 DOI: 10.1529/biophysj.108.133496] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Perfluorocarbons (PFCs) hold great promise for biomedical applications. However, relatively little is known about the impact of these chemicals on membranes. We used unilamellar vesicles to explore the effects of PFCs on membrane packing and vesicle stability. Four clinically relevant PFCs with varying vapor pressures (PP1, 294 mbar; PP2, 141 mbar; PP4, 9.6 mbar; and PP9, 2.9 mbar) were examined. Microscopy imaging and spectroscopic measurements suggest that PFCs, especially those with high vapor pressures, lead to vesicle fusion within hours. Upon exposure to PP1 and PP2 for 72 h, vesicles retained a spherical shape, but the size changed from approximately 200 nm to approximately 20-40 mum. In addition, membrane packing underwent marked changes during this timeframe. A significant decrease in water content in the lipid polar headgroup regions occurred during the first 1-2-h exposure to PFCs, followed by a steady increase in water content over time. Possible mechanisms were proposed to explain these dramatic structural changes. The finding that chemically inert PFCs exhibited fusogenic activity and marked changes in membrane surface packing is novel, and should be considered when using PFCs for biomedical applications.
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Korashy HM, El-Kadi AOS. NF-κB and AP-1 are key signaling pathways in the modulation of NAD(P)H:Quinone oxidoreductase 1 gene by mercury, lead, and copper. J Biochem Mol Toxicol 2008; 22:274-83. [DOI: 10.1002/jbt.20238] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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Ding T, Li Z, Hailemariam T, Mukherjee S, Maxfield FR, Wu MP, Jiang XC. SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis. J Lipid Res 2008; 49:376-85. [DOI: 10.1194/jlr.m700401-jlr200] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
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Guilbault C, De Sanctis JB, Wojewodka G, Saeed Z, Lachance C, Skinner TAA, Vilela RM, Kubow S, Lands LC, Hajduch M, Matouk E, Radzioch D. Fenretinide Corrects Newly Found Ceramide Deficiency in Cystic Fibrosis. Am J Respir Cell Mol Biol 2008; 38:47-56. [PMID: 17656682 DOI: 10.1165/rcmb.2007-0036oc] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Chronic and persistent lung infections cause the majority of morbidity and mortality in patients with cystic fibrosis (CF). Galactosyl ceramide has been previously shown to be involved in Pseudomonas internalization. Therefore, we assessed ceramide levels in the plasma of patients with CF and compared them to healthy volunteers using high-performance liquid chromatography followed by mass spectrometry. Our results demonstrate that patients with CF display significantly lower levels of several ceramide sphingolipid species, specifically C14:0, C20:1, C22:0, C22:1, and C24:0 ceramides, and dihydroxy ceramide (DHC16:0). We report that Cftr-knockout mice display diminished ceramide levels in CF-related organs (lung, pancreas, ileum, and plasma) compared with their littermate controls. Since it has been previously reported that in vitro treatment with fenretinide induced ceramide in neuroblastoma cell lines, we decided to test this drug in vivo using our Cftr-knockout mice in an attempt to correct this newly identified defect in ceramide levels. We demonstrate that treatment with fenretinide is able to increase ceramide concentrations in CF-related organs. We further assessed the biological effect of fenretinide on the ability of Cftr-knockout mice to combat lung infection with P. aeruginosa. Our data show dramatic improvement in the ability of Cftr-knockout mice to control P. aeruginosa infection. Overall, these findings not only document a novel deficiency in several ceramide species in patients with CF, but also demonstrate a pharmacologic means to correct this defect in Cftr-knockout mice. Our data provide a strong rationale for clinical intervention that may benefit patients with CF suffering from CF lung disease.
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Affiliation(s)
- Claudine Guilbault
- McGill University Health Center, Montreal General Hospital Research Institute, 1650 Cedar Avenue, Room L11-218, Montreal, PQ, H3G 1A4 Canada
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Takahashi H, Namiki H. Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors. Biochem J 2007; 405:331-40. [PMID: 17373912 PMCID: PMC1904528 DOI: 10.1042/bj20070299] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.
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Affiliation(s)
- Hideyuki Takahashi
- Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169-0051, Japan.
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Vilela RM, Lands LC, Meehan B, Kubow S. Inhibition of IL-8 release from CFTR-deficient lung epithelial cells following pre-treatment with fenretinide. Int Immunopharmacol 2006; 6:1651-64. [PMID: 16979119 DOI: 10.1016/j.intimp.2006.06.012] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2006] [Revised: 06/20/2006] [Accepted: 06/21/2006] [Indexed: 11/15/2022]
Abstract
Cystic fibrosis (CF) is characterized by a biochemical abnormality in the cystic fibrosis transmembrane conductance regulator (CFTR) channel. CFTR-deficient lung epithelial cells may have high constitutive glutathione (GSH) levels that could decrease the intracellular content of the sphingolipid second messenger, ceramide. Altered ceramide levels in CF cells could, in turn, lead to their resistance to apoptosis and an immune hyper-responsiveness. As fenretinide is a ceramide up-regulating drug that inhibits the activation of the pro-inflammatory transcriptional factor, nuclear factor (NF)-kappaB, the impact of fenretinide on unstimulated and tumor necrosis factor (TNF)-alpha stimulated production of NF-kappaB-dependent interleukin (IL)-8 was studied in immortalized wild-type (non-CF; 9HTEo-) and mutant DeltaF508 CFTR (CF; CFTE29o-) tracheal epithelial cells. Despite higher constitutive levels of GSH in CF cells, their intracellular ceramide content showed a greater enhancement following fenretinide and TNF-alpha treatment than non-CF cells. Clinically relevant concentrations of fenretinide (1.25, 2.5 and 5 microM) inhibited TNF-alpha-induced IL-8 production of CF cells by up to 73% but had no effect or increased the IL-8 production in non-CF cells. Although fenretinide treatment was associated with a higher intracellular ceramide content in the mutant DeltaF508 CFTR cells, the fenretinide-mediated decrease in IL-8 secretion was not consistently explained by changes in the intracellular content of this sphingolipid. Fenretinide was ineffective in increasing the susceptibility to apoptosis in CF cells whereas non-CF cells were sensitive to the apoptosis induced by both fenretinide and cisplatin exposure. The fenretinide mediated decrease in IL-8 release in CF cells under TNF-alpha stimulated conditions presents the possibility that the lung inflammation in CF could be attenuated via low dose fenretinide treatment.
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Affiliation(s)
- Regina Maria Vilela
- School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, 21,111 Lakeshore, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9
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Abstract
AIM: To investigate the effect of exogenous ceramide-induced apoptosis on human colon carcinoma HT-29 cells.
METHODS: Light microscope, transmission electron microscope and fluorescence microscope were used to observe the morphology change of apoptosis in HT-29 cells. Agarose gel electrophoresis was performed to detect the DNA fragment. Mitochondrial function was detected by MTT assay. mRNA expression of Bcl-2 family gene members was determined by reverse transcription polymerase chain reaction (RT-PCR) assay.
RESULTS: After C2-ceramide treatment, typical characteristics of apoptosis, such as nuclear chromatin breakage, apoptotic body and DNA ladder, could be observed. After exposure to 50 μmol/L C2-ceramide for 12 and 24 h, cell apoptosis was 64.1% and 81.3% respectively, which had a time-and dose-effect relationship. Mitochondrial function started to decrease from 6 h after exposure to ceramide. Meanwhile, ceramide up-regulated or down-regulated the mRNA expression of Bcl-2 family gene members.
CONCLUSION: Ceramide induces apoptosis of human colon carcinoma HT-29 cells by affecting the expression of Bcl-2 family gene members and impacting the mitochondrial function.
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Affiliation(s)
- Xiao-Feng Zhang
- Department of Toxicology, Public Health College, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081, Heilongjiang Province, China
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Wang C, Li Y, Xiong J, Tan Y, Yu J. Using of the surface plasmon resonance cytosensor for real-time and non-invasive monitoring of cellular effects in living C6 cells induced by PMA. ACTA ACUST UNITED AC 2006. [DOI: 10.1007/s11434-006-0927-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
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Kim WH, Choi CH, Kang SK, Kwon CH, Kim YK. Ceramide induces non-apoptotic cell death in human glioma cells. Neurochem Res 2006; 30:969-79. [PMID: 16258846 DOI: 10.1007/s11064-005-6223-y] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/02/2005] [Indexed: 10/25/2022]
Abstract
Ceramide causes either apoptosis or non-apoptotic cell death depending on model system and experimental conditions. The present study was undertaken to examine the effect of ceramide on cell viability and its molecular events leading to cell death in A172 human glioma cells. Ceramide induced cell death in a dose-dependent manner and the cell death was dependent on generation of reactive oxygen species and lipid peroxidation. TUNEL assay, Hoechst 33258 staining, and flow cytometric analysis did not show typical apoptotic morphological features. Ceramide caused phosphorylation of extracellular signal-regulated kinase (ERK) and p38, but the cell death was not affected by inhibitors of MAPK subfamilies. Ceramide caused ATP depletion without loss of mitochondrial membrane potential. Ceramide did not induce caspase activation and ceramide-induced cell death was also not altered by inhibitors of caspase activation. Transfection of dominant inhibitory mutant of IkappaBalpha (S32A/36A) and pretreatment of pyrrolidinedithiocarbamate, an inhibitor of NF-kappaB, enhanced ceramide-induced cell death. These results indicate that ceramide causes non-apoptotic, caspase-independent cell death by inducing reactive oxygen species generation in A172 human glioma cells. NF-kappaB is involved in the regulation of ceramide-induced cell death in human glioma cells.
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Affiliation(s)
- Wi Hyun Kim
- Department of Neurosurgery, College of Medicine, Pusan National University, 602-739, Pusan, Korea
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21
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Devidze N, Pfaff DW, Kow LM. Potentiation of genomic actions of estrogen by membrane actions in mcf-7 cells and the involvement of protein kinase C activation. Endocrine 2005; 27:253-8. [PMID: 16230781 DOI: 10.1385/endo:27:3:253] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/06/2005] [Revised: 06/02/2005] [Accepted: 06/10/2005] [Indexed: 02/05/2023]
Abstract
It is now well established that estrogens (E) have at least two kinds of actions: genomic and nongenomic. But the relationship between these actions has hardly been explored. In this study we investigated this relationship in MCF-7 cells, a human breast cancer cell line, and explored the possible involvement of protein kinase C (PKC) signaling pathways. For this purpose a two-pulse paradigm was used: cells were treated with 17beta-estradiol (E), E conjugated with bovine serum albumin (E-BSA or fE'), or other test agents in the first pulse and with E in the second pulse following a 4-h interval. An E-BSA+E paradigm was used to show that replacement of E with the membrane-impermeable E-BSA in the first pulse could potentiate genomic actions of E, in the second pulse. To investigate involvement of signaling pathways, two PKC activators, phorbol 12,13-diacetate (PDAc) or phorbol 12-myristate 13-acetate (PMA), and inhibitors (chelerythrine chloride and H7-dihydrochloride) were used to replace E or E-BSA in the first pulse. PDAc was as effective as E or E-BSA in potentiating the genomic action of E in the second pulse, while PMA was almost without an effect. Conversely, the potentiating effects of E-BSA and PDAc were blocked by chelerythrine chloride but, interestingly, not by H7. The exact reason underlying these differences is not known. In summary, in MCF-7 cells a membrane action of E can potentiate a later genomic action and involves PKC signaling.
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Affiliation(s)
- Nino Devidze
- Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, NY 10021, USA.
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22
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Claus RA, Wüstholz A, Müller S, Bockmeyer CL, Riedel NH, Kinscherf R, Deigner HP. Synthesis and Antiapoptotic Activity of a Novel Analogue of the Neutral Sphingomyelinase Inhibitor Scyphostatin. Chembiochem 2005; 6:726-37. [PMID: 15751001 DOI: 10.1002/cbic.200400228] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The enantioselective synthesis of an analogue of scyphostatin, a potent inhibitor of the neutral sphingomyelinase, is described. The synthesis starts with cyclohexanone and a protected D-serine derivative. The key step is an asymmetric hydroxylation to access a hydroxycyclohexanone, which is transformed into a substituted hydroxycyclohexenone. This is converted into the scyphostatin analogue 14, a chemically and metabolically stabilised compound lacking the epoxy function of the natural congener and carrying a palmitic acid group instead of the native trienoyl residue. An evaluation of the biological activity of 14 revealed neutral sphingomyelinase inhibition in several in vivo test systems (monocytes, macrophages, hepatocytes) monitoring antiapoptotic effects and the inversion of phorbolester-induced translocation of green fluorescent protein labelled kinase (protein kinase C-alpha).
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Affiliation(s)
- Ralf A Claus
- Department for Anaesthesiology and Intensive Care Therapy, Division for Experimental Anaesthesiology, Friedrich-Schiller-University Jena, Research Centre Lobeda, Erlanger Allee 101, 07747 Jena, Germany
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23
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Becker KP, Kitatani K, Idkowiak-Baldys J, Bielawski J, Hannun YA. Selective Inhibition of Juxtanuclear Translocation of Protein Kinase C βII by a Negative Feedback Mechanism Involving Ceramide Formed from the Salvage Pathway. J Biol Chem 2005; 280:2606-12. [PMID: 15546881 DOI: 10.1074/jbc.m409066200] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
In a previous study, we showed that protein kinase C betaII (PKC betaII) translocated to a novel juxtanuclear compartment as observed in several cell types (Becker, K. P., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 52747-52754). In this study, we noted the absence of this translocation in MCF-7 breast cancer cells, and we examined the mechanisms underlying this selectivity of response. We show that sustained stimulation of PKC betaII with 4beta-phorbol 12-myristate 13-acetate (PMA) resulted in accumulation of ceramide in MCF-7 cells but not in those cells that showed juxtanuclear translocation of PKC betaII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC betaII in HEK 293 cells. On the other hand, inhibition of ceramide accumulation with fumonisin B1 restored the ability of PMA to induce translocation of PKC betaII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC betaII in response to PMA. Investigation of the mechanisms of ceramide generation in response to PMA revealed that PMA activated the salvage pathway of ceramide formation and not the de novo pathway. This conclusion was based on the following: 1) the ability of fumonisin B1 but not myriocin to inhibit ceramide formation, 2) the ability of PMA to induce increases in palmitate-labeled ceramide only under chase labeling but not acute pulse labeling, 3) the induction of the levels of sphingosine but not dihydrosphingosine in response to PMA, and 4) induction of sphingomyelin hydrolysis in response to PMA. Together, these results define a novel pathway of regulated formation of ceramide, the salvage pathway, and they define a role for this pathway in regulating juxtanuclear translocation of PKC betaII.
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Affiliation(s)
- Kevin P Becker
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA
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24
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Abboushi N, El-Hed A, El-Assaad W, Kozhaya L, El-Sabban ME, Bazarbachi A, Badreddine R, Bielawska A, Usta J, Dbaibo GS. Ceramide inhibits IL-2 production by preventing protein kinase C-dependent NF-kappaB activation: possible role in protein kinase Ctheta regulation. THE JOURNAL OF IMMUNOLOGY 2004; 173:3193-200. [PMID: 15322180 DOI: 10.4049/jimmunol.173.5.3193] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The role of the sphingolipid ceramide in modulating the immune response has been controversial, in part because of conflicting data regarding its ability to regulate the transcription factor NF-kappaB. To help clarify this role, we investigated the effects of ceramide on IL-2, a central NF-kappaB target. We found that ceramide inhibited protein kinase C (PKC)-mediated activation of NF-kappaB. Ceramide was found to significantly reduce the kinase activity of PKCtheta as well as PKCalpha, the critical PKC isozymes involved in TCR-induced NF-kappaB activation. This was followed by strong inhibition of IL-2 production in both Jurkat T leukemia and primary T cells. Exogenous sphingomyelinase, which generates ceramide at the cell membrane, also inhibited IL-2 production. As expected, the repression of NF-kappaB activation by ceramide led to the reduction of transcription of the IL-2 gene in a dose-dependent manner. Inhibition of IL-2 production by ceramide was partially overcome when NF-kappaB nuclear translocation was reconstituted with activation of a PKC-independent pathway by TNF-alpha or when PKCtheta was overexpressed. Importantly, neither the conversion of ceramide to complex glycosphingolipids, which are known to have immunosuppressive effects, nor its hydrolysis to sphingosine, a known inhibitor of PKC, was necessary for its inhibitory activity. These results indicate that ceramide plays a negative regulatory role in the activation of NF-kappaB and its targets as a result of inhibition of PKC.
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Affiliation(s)
- Nour Abboushi
- Department of Biochemistry, American University of Beirut, Faculty of Medicine, Lebanon
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25
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Takahashi H, Suzuki K, Namiki H. Pervanadate-induced reverse translocation and tyrosine phosphorylation of phorbol ester-stimulated protein kinase C betaII are mediated by Src-family tyrosine kinases in porcine neutrophils. Biochem Biophys Res Commun 2004; 314:830-7. [PMID: 14741711 DOI: 10.1016/j.bbrc.2003.12.163] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Protein kinase C (PKC), upon activation, translocates from the cytosol to the plasma membrane. Phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, is known to induce irreversible translocation of PKC to the plasma membrane, in contrast to the reversible translocation resulting from physiological stimuli and subsequent rapid return to the cytosol (reverse translocation). However, we have previously shown that tyrosine phosphatase (PTPase) inhibitors induce reverse translocation of PMA-stimulated PKCbetaII in porcine polymorphonuclear leukocytes (PMNs). In the present study, we showed that pervanadate, a potent PTPase inhibitor, also induces tyrosine phosphorylation of PMA-stimulated PKCbetaII in porcine PMNs. Furthermore, PP2, a specific inhibitor of Src-family tyrosine kinases (PTKs), was found to inhibit both pervanadate-induced reverse translocation and tyrosine phosphorylation of PMA-stimulated PKCbetaII, suggesting that these two pervanadate-induced responses are mediated by Src-family PTKs. Our findings provide novel insight into the relationship between the subcellular localization and tyrosine phosphorylation of PKC.
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Affiliation(s)
- Hideyuki Takahashi
- Department of Biology, School of Education, Waseda University, Shinjuku-ku, 169-0051, Tokyo, Japan
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Heung LJ, Luberto C, Plowden A, Hannun YA, Del Poeta M. The sphingolipid pathway regulates Pkc1 through the formation of diacylglycerol in Cryptococcus neoformans. J Biol Chem 2004; 279:21144-53. [PMID: 15014071 DOI: 10.1074/jbc.m312995200] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The sphingolipid biosynthetic pathway generates bioactive molecules crucial to the regulation of mammalian and fungal physiological and pathobiological processes. In previous studies (Luberto, C., Toffaletti, D. L., Wills, E. A., Tucker, S. C., Casadevall, A., Perfect, J. R., Hannun, Y. A., and Del Poeta, M. (2001) Genes Dev. 15, 201-212), we demonstrated that an enzyme of the fungal sphingolipid pathway, Ipc1 (inositol-phosphorylceramide synthase-1), regulates melanin, a pigment required for the pathogenic fungus Cryptococcus neoformans to cause disease. In this study, we investigated the mechanism by which Ipc1 regulates melanin production. Because Ipc1 also catalyzes the production of diacylglycerol (DAG), a physiological activator of the classical and novel isoforms of mammalian protein kinase C (PKC), and because it has been suggested that PKC is required for melanogenesis in mammalian cells, we investigated whether Ipc1 regulates melanin in C. neoformans through the production of DAG and the subsequent activation of Pkc1, the fungal homolog of mammalian PKC. The results show that modulation of Ipc1 regulates the levels of DAG in C. neoformans cells. Next, we demonstrated that C. neoformans Pkc1 is a DAG-activated serine/threonine kinase and that the C1 domain of Pkc1 is necessary for this activation. Finally, through both pharmacological and genetic approaches, we found that inhibition of Pkc1 abolishes melanin formation in C. neoformans. This study identifies a novel signaling pathway in which C. neoformans Ipc1 plays a key role in the activation of Pkc1 through the formation of DAG. Importantly, this pathway is essential for melanin production with implications for the pathogenicity of C. neoformans.
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Affiliation(s)
- Lena J Heung
- Department of Biochemistry, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA
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27
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Matthews V, Schuster B, Schütze S, Bussmeyer I, Ludwig A, Hundhausen C, Sadowski T, Saftig P, Hartmann D, Kallen KJ, Rose-John S. Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE). J Biol Chem 2003; 278:38829-39. [PMID: 12832423 DOI: 10.1074/jbc.m210584200] [Citation(s) in RCA: 294] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
Interleukin-6 (IL-6) activates cells by binding to the membrane-bound IL-6 receptor (IL-6R) and subsequent formation of a glycoprotein 130 homodimer. Cells that express glycoprotein 130, but not the IL-6R, can be activated by IL-6 and the soluble IL-6R which is generated by shedding from the cell surface or by alternative splicing. Here we show that cholesterol depletion of cells with methyl-beta-cyclodextrin increases IL-6R shedding independent of protein kinase C activation and thus differs from phorbol ester-induced shedding. Contrary to cholesterol depletion, cholesterol enrichment did not increase IL-6R shedding. Shedding of the IL-6R because of cholesterol depletion is highly dependent on the metalloproteinase ADAM17 (tumor necrosis factor-alpha-converting enzyme), and the related ADAM10, which is identified here for the first time as an enzyme involved in constitutive and induced shedding of the human IL-6R. When combined with protein kinase C inhibition by staurosporine or rottlerin, breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide also increased IL-6R shedding. The effect of cholesterol depletion was confirmed in human THP-1 and Hep3B cells and in primary human peripheral blood monocytes, which naturally express the IL-6R. For decades, high cholesterol levels have been considered harmful. This study indicates that low cholesterol levels may play a role in shedding of the membrane-bound IL-6R and thereby in the immunopathogenesis of human diseases.
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Affiliation(s)
- Vance Matthews
- Biochemisches Institut, Christian Albrechts Universität zu Kiel, Germany
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Tisdale EJ, Wang J, Silver RB, Artalejo CR. Atypical protein kinase C plays a critical role in protein transport from pre-Golgi intermediates. J Biol Chem 2003; 278:38015-21. [PMID: 12871960 DOI: 10.1074/jbc.m305381200] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
The small GTPase Rab2 requires atypical protein kinase C iota/lambda (PKCiota/lambda) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702-712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCiota/lambda in the early secretory pathway. A peptide corresponding to the unique PKCiota/lambda pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCiota/lambda, beta-COP, and p53/p58. Because Rab2, beta-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCiota/lambda (W274K). Both the pseudosubstrate peptide and kinase-dead PKCiota/lambda in tandem with Rab2 caused sustained membrane association of PKCiota/lambda, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCiota/lambda was reversed by PKCiota/lambda wild type. These combined results indicate that PKCiota/lambda is essential for protein transport in the early secretory pathway and suggest that PKCiota/lambda kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.
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Affiliation(s)
- Ellen J Tisdale
- Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
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Takahashi H, Suzuki K, Namiki H. Phenylarsine oxide and H2O2 plus vanadate induce reverse translocation of phorbol-ester-activated PKCbetaII. Cell Struct Funct 2003; 28:123-30. [PMID: 12808232 DOI: 10.1247/csf.28.123] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
The intracellular localization of protein kinase C (PKC) is important for the regulation of its biological activity. Recently, it was reported that, whereas phorbol esters such as PMA induce prolonged translocation of PKC to the plasma membrane, with physiological stimuli, the translocation of PKC is transient and followed by rapid return to the cytoplasm. In addition, this membrane dissociation of PKC was shown to require both the kinase activity of PKC and the phosphorylation of its carboxyl terminus autophosphorylation sites. However, the detailed molecular mechanism of PKC reverse translocation remains obscure. We demonstrated that in porcine polymorphonuclear leucocytes (PMNs), phenylarsine oxide (PAO), a putative protein tyrosine phosphatase (PTPase) inhibitor, induced reverse translocation of PMA-stimulated PKCbetaII. Hydrogen peroxide (H(2)O(2)) in combination with vanadate, both of which are PTPase inhibitors, also induced reverse translocation of PKCbetaII. H(2)O(2) or vanadate alone had little effect on PMA-induced PKCbetaII translocation. Furthermore, genistein and ethanol, which are inhibitors of tyrosine kinase and phospholipase D, respectively, prevented the PKCbetaII reverse translocation induced by the PTPase inhibitors. These results indicate, for the first time, that the tyrosine phosphorylation/phospholipase D pathway may be involved in the process of membrane dissociation of PKC.
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Affiliation(s)
- Hideyuki Takahashi
- Department of Biology, School of Education, Waseda University, Tokyo 169-0051, Japan
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Abstract
A model for the possible involvement of Protein Kinase C (PKC) in the pathogenesis of inborn errors of metabolism has been proposed. According to this model, perturbation of PKC activity by the accumulation of naturally occurring compounds serves as a unifying functional link between genotype and phenotype. Recent reports regarding an increasing number of modulating metabolites, specific PKC-subtypes activities, their effect on transcription factors and gene expression in various diseases and additional PKC-substrates expand the model. A re-examination of the proposed model in view of these reports and, vice versa, a review of these reports in the context of the proposed model reveal some common phenotypic outcomes in inborn errors of fatty acid-, cholesterol- and homocystine-metabolism as well as lysosomal and peroxisomal diseases.
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Affiliation(s)
- Avihu Boneh
- Metabolic Service, Genetic Health Services, Victoria, Australia.
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