Increased Levels of Inositol Hexakisphosphate (InsP6) Protect HEK293 Cells from Tumor Necrosis Factor α- and Fas-induced Apoptosis

The overexpression of inositol 1,3,4-trisphosphate 5/6-kinase has recently been shown to protect HEK293 cells from tumor necrosis factor α (TNFα)-induced apoptosis. This overexpression leads to an increase in the levels of both inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol 1,2,3,4,5,6-hexakisphosphate (InsP6). Cells that overexpress InsP5 2-kinase have increased levels of InsP6 and are also protected from TNFα-induced apoptosis; furthermore, cells that express an RNA interference construct to the 2-kinase are deficient in InsP6 and are sensitized to TNFα-induced apoptosis. Therefore the protective effect of 5/6-kinase on TNFα-mediated apoptosis is due to an increase of InsP6 or to a metabolite derived from InsP6. Furthermore, we find that the InsP6 also protects from Fas-mediated apoptosis. No effect was seen in the endocytic rate of transferrin receptor, caspase 8 activity, or TNF receptor number at the cell surface. Cells that overexpress 2-kinase do show an increase in the amount of receptor-interacting protein (RIP), while cells with reduced InsP6 levels show relatively less RIP, providing a possible mechanism for the effect on apoptosis. The overexpression of inositol 1,3,4-trisphosphate 5/6-kinase has recently been shown to protect HEK293 cells from tumor necrosis factor α (TNFα)-induced apoptosis. This overexpression leads to an increase in the levels of both inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol 1,2,3,4,5,6-hexakisphosphate (InsP6). Cells that overexpress InsP5 2-kinase have increased levels of InsP6 and are also protected from TNFα-induced apoptosis; furthermore, cells that express an RNA interference construct to the 2-kinase are deficient in InsP6 and are sensitized to TNFα-induced apoptosis. Therefore the protective effect of 5/6-kinase on TNFα-mediated apoptosis is due to an increase of InsP6 or to a metabolite derived from InsP6. Furthermore, we find that the InsP6 also protects from Fas-mediated apoptosis. No effect was seen in the endocytic rate of transferrin receptor, caspase 8 activity, or TNF receptor number at the cell surface. Cells that overexpress 2-kinase do show an increase in the amount of receptor-interacting protein (RIP), while cells with reduced InsP6 levels show relatively less RIP, providing a possible mechanism for the effect on apoptosis. The pathways that produce the higher soluble inositol phosphates in human cells have been elucidated recently (1Verbsky J.W. Wilson M.P. Kisseleva M.V. Majerus P.W. Wente S.R. J. Biol. Chem. 2002; 277: 31857-31862Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 2Chang S.C. Miller A.L. Feng Y. Wente S.R. Majerus P.W. J. Biol. Chem. 2002; 277: 43836-43843Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 3Wilson M.P. Majerus P.W. J. Biol. Chem. 1996; 271: 11904-11910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The action of phospholipase C on phosphatidylinositol 4,5-bisphosphate, yields inositol 1,4,5-trisphosphate (InsP3) 1The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; Ins(1,3,4)P3, inositol 1,3,4-trisphosphate; Ins(1,3,4,6)P4, inositol 1,3,4,6-tetrakisphosphate; InsP5, inositol 1,3,4,5,6-pentakisphosphate; InsP6, inositol 1,2,3,4,5,6-hexakisphosphate; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PARP, poly(ADP-ribose) polymerase; cPARP, cleaved PARP; TNF, tumor necrosis factor; RIP, receptor-interacting protein; RNAi, RNA interference. and diacyl-glycerol. In mammalian cells, InsP3 is phosphorylated to inositol 1,3,4,5-tetrakisphosphate by an InsP3 3-kinase and dephosphorylated to inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) by a 5-phosphatase (4Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (349) Google Scholar). Ins(1,3,4)P3 is then phosphorylated to inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by the Ins(1,3,4)P3 5/6-kinase, to inositol 1,3,4,5,6-pentakisphosphate (InsP5) by Ins(1,3,4,6)P4 5-kinase, and to inositol 1,2,3,4,5,6-hexakisphosphate (InsP6) by InsP5 2-kinase (1Verbsky J.W. Wilson M.P. Kisseleva M.V. Majerus P.W. Wente S.R. J. Biol. Chem. 2002; 277: 31857-31862Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 5Fujii M. York J.D. J. Biol. Chem. 2005; 280: 1156-1164Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In cell culture, production of InsP6 is regulated by the activity of 5/6-kinase, which is rate-limiting (6Verbsky J.W. Chang S.C. Wilson M.P. Mochizuki Y. Majerus P.W. J. Biol. Chem. 2005; 280: 1911-1920Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) producing the committed isomer in the synthesis of InsP6, Ins(1,3,4,6)P4. Overexpression of 5/6-kinase results in an increase in InsP5 and InsP6, while depletion of 5/6-kinase results in the loss of InsP5 and InsP6. Another function for the inositol (1,3,4)P3 5/6-kinase other than phosphorylating inositol phosphates was recently discovered. Wilson et al. (7Wilson M.P. Sun Y. Cao L. Majerus P.W. J. Biol. Chem. 2001; 276: 40998-41004Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) have shown that 5/6-kinase co-purifies with the COP9 signalosome from cow brain. This complex of nine proteins has been shown to have the ability to phosphorylate c-Jun, IκBα, and p53 (8Naumann M. Bech-Otschir D. Huang X. Ferrell K. Dubiel W. J. Biol. Chem. 1999; 274: 35297-35300Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 9Bech-Otschir D. Kraft R. Huang X. Henklein P. Kapelari B. Pollmann C. Dubiel W. EMBO J. 2001; 20: 1630-1639Crossref PubMed Scopus (328) Google Scholar, 10Seeger M. Kraft R. Ferrell K. Bech-Otschir D. Dumdey R. Schade R. Gordon C. Naumann M. Dubiel W. FASEB J. 1998; 12: 469-478Crossref PubMed Scopus (313) Google Scholar). Wilson et al. (7Wilson M.P. Sun Y. Cao L. Majerus P.W. J. Biol. Chem. 2001; 276: 40998-41004Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) subsequently showed that 5/6-kinase purified from insect cells also phosphorylates c-Jun, p53, and IκBα, making it likely that in part the protein kinase activity of the COP9 signalosome may be attributed to 5/6-kinase. c-Jun and IκB are both involved in TNFα signaling and apoptosis. TNFα is involved in numerous processes including cell death and development and oncogenesis and immune, inflammatory, and stress responses (11Chen G.Q. Goeddel D.V. Science. 2002; 296: 1634-1635Crossref PubMed Scopus (1505) Google Scholar). TNFα acts in opposing ways with regards to apoptosis. Through one arm of the pathway it can activate transcription by NFκB; TRADD is recruited to the TNF receptor through its death domains, and it in turn recruits RIP, a death domain-containing kinase, and TRAF2. Together TRAF2 and RIP recruit and activate IκB kinase complex, which phosphorylates IκBα, signaling it for ubiquitination and destruction. NFκB is then translocated to the nucleus to stimulate transcription. Two of the many transcriptional targets of NFκB are FLIP and cIAP, proteins that inhibit apoptosis, and thus NFκB activity is considered anti-apoptotic. The second of the pathways it shares with Fas; this pathway stimulates apoptosis by recruiting FADD and caspase 8, which results in the cleavage and activation of caspase 8, initiating the caspase cascade. The pro-apoptotic action of TNFα cannot overcome its anti-apoptotic activity through NFκB in most cells; thus, to induce apoptosis in cells, protein synthesis has to be inhibited or NFκB signaling has to be blocked. The ability of 5/6-kinase to phosphorylate proteins involved in TNFα-mediated apoptosis led Sun et al. (12Sun Y. Mochizuki Y. Majerus P.W. J. Biol. Chem. 2003; 278: 43645-43653Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) to determine whether the overexpression of 5/6-kinase had an effect on apoptosis. They found that HEK293 cells were protected from TNFα-induced apoptosis when there were elevated amounts of 5/6-kinase. They investigated whether this protection was due to increased NFκB signaling, but they found no difference in IκBα stability or NFκB activity by gel shifts of 5/6-kinase-expressing lines. This led to the possibility that the protection from apoptosis was due to inhibition of caspase activation rather than NFκB stimulation. It also suggested that the protection from apoptosis may not be due to the associated protein kinase activity of 5/6-kinase but rather through its inositol phosphate kinase activity and thus to the soluble, more highly phosphorylated inositol phosphates. We recently have shown that an increase in 5/6-kinase activity in cells results in a concomitant increase of InsP5 and InsP6. Thus activities ascribed to the inositol phosphate kinase function of 5/6-kinase may be due to actions of InsP5 or InsP6 or to another downstream metabolite. Here we show that expression of 2-kinase and an increase of InsP6 can protect cells from TNFα-mediated apoptosis, and we attribute the protective effect of 5/6-kinase on TNFα-mediated apoptosis to the production of InsP6 itself. In addition, we find that expression of 2-kinase also results in a protection from Fas mediated apoptosis, and thus InsP6 may be a general regulator of apoptosis. Furthermore, we notice that altered InsP6 levels in cells result in altered levels of the protein RIP, which may provide a mechanism for the role of InsP6 in apoptosis. All chemicals were reagent grade or better. Restriction endonucleases, DNA modifying enzymes, and general reagents were from Amersham Biosciences, Roche Applied Sciences, Fisher, Invitrogen, New England Biolabs, Promega Corp., Sigma, and Stratagene unless stated otherwise. Acrylamide solution, Bio-Safe Coomassie Blue stain, and Bradford protein assay kit used for protein work were purchased from Bio-Rad. The SuperSignal West Pico kit used for detection of Western transfer blots was from Pierce. Radiolabeled [3H]inositol and [3H]InsP6 were purchased from American Radiolabeled Chemicals (St. Louis, MO) and Amersham Biosciences, respectively. TNFα was obtained from Peprotech and activating Fas antibody from BD Biosciences. Anti-caspase 8 (c20) goat polyclonal antibody was obtained from Santa Cruz Biotechnology, anti-caspase 8 monoclonal antibody (mAb) from Cell Signaling, and anti-RIP mAb from BD Biosciences. Protein G was obtained from Sigma. Strains, Plasmids, and Growth Conditions—Methods for Escherichia coli growth and selection were described previously (13Chanda V.B. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1997Google Scholar, 14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). E. coli strain XL-1Blue (Stratagene) was used as the bacterial host for all plasmids unless stated otherwise. Bacterial strains were cultured in LB (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) medium supplemented with ampicillin (100 μg/ml) where appropriate and transformed by standard methods. All bacterial strains were grown at 37 °C Cloning, Production, and Maintenance of Cell Lines—Stable cell lines expressing 2-kinase were reported previously (6Verbsky J.W. Chang S.C. Wilson M.P. Mochizuki Y. Majerus P.W. J. Biol. Chem. 2005; 280: 1911-1920Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The stably transfected 2-kinase RNAi line was produced as follows. Oligonucleotides containing the antisense target to the 2-kinase (5′-GAAGACCTCGGAAGAGATA-3′) or to luciferase (5′-CTTACGCTGAGTACTTCGA-3′) were annealed and ligated in the pSUPER vector (a gift from Dr. Reuven Agami). Equal amounts of either the 2-kinase or the luciferase RNAi construct were mixed 7:1 with pBabe containing a puromycin resistance gene and transfected into the same parent cells as the overexpression lines, TRex HEK293(Invitrogen), using Lipofectamine as per the manufacturer's protocol. Cells were allowed to recover for 1 day and serially diluted, and clones were selected with 1 μg/ml puromycin. Individual clones were obtained and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1 μg/ml puromycin. High Performance Liquid Chromatography (HPLC)—HPLC was conducted as follows. Cell were grown in the presence of [3H]inositol (10 μCi/ml) for 4 days. Cells were lysed in methanol/0.5 n HCl (2:1) and extracted with chloroform. The aqueous phase was separated, dried, and resuspended in distilled water. Samples were applied to a Whatman Partisphere SAX strong anion exchange column (4.6 mm x125 mm) running a 30 min gradient from 0 to 1.7 m ammonium phosphate, pH 3.5, followed by a 30-min isocratic elution with 1.7 m ammonium phosphate, pH was using the and the of the inositol phosphates was on the of with Cell were grown on to then with 1 TNFα and μg/ml) for cells were using the kit Western cells were from the by with PBS, and lysed in mm pH mm NaCl, 10% Roche Applied Cells were to in an and was by at in an at 4 The protein of the was using the Bradford assay protein as per the manufacturer's protocol. standard of protein of was a 10% gel for and subsequently Western was conducted using polyclonal antibody using standard The appropriate antibody and the SuperSignal West Pico were used to the appropriate were by of Western blots using an and the were using TNFα or Fas of HEK293 cells were in in the presence the lines or vector or 2-kinase RNAi or luciferase of for and then with TNFα μg/ml) or the stated amount of Fas antibody protein G (a of Fas to protein for the amount of were for Western blots as described RNAi in cells lines were transfected with the same 2-kinase RNAi construct used to cell lines, grown for 1 day to then into and grown for in the presence of to induce 5/6-kinase cells were then for with TNFα and as were with μg/ml) in medium bovine serum in Dulbecco's modified Eagle's at 37 °C for the of the the medium was and the were with to The cells were then for min with m pH containing m at 4 The was with a in the same and used to determine the amount of The cells were lysed in 1 n to determine the The of to was a of was and the cells were as to determine the caspase 8 as were used for Cells were in and lysed in of mm pH mm NaCl, 10% for 30 min on of was on protein for 1 and the caspase 8 complex was with of anti-caspase 8 antibody and of protein at 4 °C were by with and to Western as that amounts of caspase 8 were of the protein was on a gel and with mAb caspase RIP was as cells were on and grown for Cells from were lysed as for the caspase 8 and RIP was with 1 of anti-RIP mAb for 1 The were with of protein for 30 min and with PBS, and the were to Western as HEK293 Cells a RNAi to the in InsP6 cell lines with a 2-kinase gene show an altered inositol phosphate as reported expression of the 2-kinase in an increase in InsP6 and a loss of We also HEK293 cells stably transfected with an RNAi construct to the a we produced cell lines transfected with the pSUPER construct containing an RNAi to the luciferase cells were with and soluble inositol phosphates purified and by HPLC, there is a of InsP6 to the by to and an increase in InsP5 and This result is with inositol of HEK293 cells transfected with to 2-kinase as reported previously (1Verbsky J.W. Wilson M.P. Kisseleva M.V. Majerus P.W. Wente S.R. J. Biol. Chem. 2002; 277: 31857-31862Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Cells to TNFα-mediated RNAi to cells show an increase of InsP5 and InsP6 and a protection from TNFα-induced apoptosis. cells show increased InsP6 and a loss of with TNFα cells show a in the number of cells, as by when with vector cells to the luciferase lines the 2-kinase RNAi lines showed more when with The results shown in were by Western Cells expressing 2-kinase and the vector were with TNFα and for and and with a polyclonal antibody PARP, a target of caspase with the results from the cell cells 2-kinase showed a in the amount of apoptosis as by the amount of cleaved to the vector lines at both We also at cleaved in 2-kinase RNAi lines. with the luciferase lines, the 2-kinase RNAi line showed more cleaved at and and is more to TNFα mediated apoptosis In both of the amount of apoptosis seen in the vector lines was than that seen in the luciferase RNAi lines. the vector lines were with the transcription was by the of as it is in the lines. The RNAi lines were not with This may for the difference the lines. it is to the increase or of apoptosis to The RNAi the from of whether the protective effect was due to the presence of InsP6 and not other from 5/6-kinase and its associated protein kinase activity, we the 2-kinase RNAi construct in cells 5/6-kinase. was an increase in apoptosis in the cells transfected with the 2-kinase RNAi to transfected with the luciferase RNAi with the we that the protective effect from TNFα-mediated apoptosis is due to the presence of InsP6 or a downstream metabolite. InsP6 while InsP6 Cells to to protection from TNFα-mediated apoptosis, we also found that 2-kinase overexpression protect cells from apoptosis as shown in Cell lines were with amounts of Fas antibody for and cell were by Western for Cells that were the 2-kinase showed less cleavage at 1 and μg/ml as with the vector lines Furthermore, the 2-kinase RNAi line showed more apoptosis as with the luciferase The results of cleavage for the μg/ml Fas were by to the of to were for RNAi and lines to the of cleaved to was in the RNAi line than luciferase line to relatively more is cleaved in the RNAi line than the luciferase in InsP6 8 or TNF it is that the protective effect of the 2-kinase on apoptosis is due to NFκB we for other that the protective is for TNF apoptosis and is mediated by a to receptor by Annu. Rev. Biochem. 2003; PubMed Scopus Google when TNF receptor is and the death signaling complex is not thus apoptosis. InsP6 has been in we transferrin in cells the seen in 2-kinase overexpression not transferrin with vector The of the was and showed no difference the 2-kinase or vector lines. Furthermore, we no difference in 2-kinase and luciferase lines not We also the TNF and Fas receptor number at the cell by and found no difference the cell lines containing increased amounts of InsP6 and with levels of InsP6. caspase 8 activity was with amounts of InsP6 or inositol to for and no effect was seen on caspase 8 activity to InsP6 or inositol Cells and RNAi of the Protein of of cell showed an increase in the of RIP in the lines as with the vector and a in the RNAi lines as with the luciferase to the RIP levels were increased by in the lines and by in the RNAi lines. The elevated amount of RIP in is in an increase in of RIP with anti-caspase The of and J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, in Scholar) was to the effect of the 2-kinase on the of the caspase complex with the TNF it was to stimulate cells with TNF to RIP with caspase 8, that the complex to a in cells with HEK293 cells more RIP protein was with caspase 8 as with the vector of cells with the the of RIP in the in the vector lines to that of the 2-kinase lines. results that in cells 2-kinase overexpression the rate of the caspase 8, and RIP The overexpression of 5/6-kinase has been shown by Sun et al. (12Sun Y. Mochizuki Y. Majerus P.W. J. Biol. Chem. 2003; 278: 43645-43653Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) to protect TNFα-mediated apoptosis, the mechanism was the overexpression of 5/6-kinase also results in an in the amounts of InsP5 and InsP6 (1Verbsky J.W. Wilson M.P. Kisseleva M.V. Majerus P.W. Wente S.R. J. Biol. Chem. 2002; 277: 31857-31862Abstract Full Text Full Text PDF PubMed Scopus (80) Google it is possible that this protective effect is due to one or more of of inositol Cells 2-kinase InsP6, InsP5, and show protection from apoptosis to vector Furthermore, HEK293 cells stably transfected with an RNAi construct to 2-kinase InsP6 and InsP5 and show an increased to apoptosis. In addition, expression of 5/6-kinase was not to overcome the to apoptosis by expression of the 2-kinase RNAi This result the of Sun et al. (12Sun Y. Mochizuki Y. Majerus P.W. J. Biol. Chem. 2003; 278: 43645-43653Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) that suggested that the protective effect of the 5/6-kinase on apoptosis is not due to its protein kinase activity but rather to its inositol kinase the protection from apoptosis is due to InsP6 or more highly phosphorylated inositol In addition, we have shown that overexpression of 2-kinase protects from apoptosis, while 2-kinase RNAi expression the cells more to apoptosis. the other in that soluble inositol phosphates are of apoptosis. results do not InsP6 in the protection from apoptosis; we can that the isomer involved has to downstream of InsP6. InsP6 is to the higher inositol and have seen no increase in an isomer when expressing the 2-kinase in cells not and there is that the inositol may stimulate apoptosis. et al. D.V. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) used an antisense to that are involved in apoptosis in an cell gene that was shown to cells to apoptosis was the inositol kinase This protein InsP6 to the acts by transcription through the the transcription of a number of targets by are involved in TNFα-mediated apoptosis or are to TNF apoptosis, InsP6 phosphorylate InsP6, expression may result in the depletion of levels of InsP6, which we show to cells to apoptosis. Thus the InsP6 and the inositol may as a apoptosis. with the InsP6 to determine the inositol isomer The protection from apoptosis by expression of the 2-kinase is not due to the anti-apoptotic of through NFκB No in IκB stability or increase in NFκB activity is seen in The protection from Fas-mediated apoptosis the role of the TNFα are in the presence of where protein synthesis is The protective effect may work on the of TNFα We a number of the possible in the activation of the of TNFα We at cell expression of the TNF receptor lines that had altered levels of InsP6 and no difference we an effect of InsP6 on caspase 8 activity in TNF activation has recently been shown to the of the TNF receptor W. J. M. J. M. M. D. Full Text Full Text PDF PubMed Scopus Google Scholar). InsP6 has been in and activation of caspase 8 by the endocytic rate of the cells with altered levels of InsP6 showed no in the of and it is that this provide the mechanism for the protection from apoptosis. Furthermore, results provide the in of the of altered InsP6 levels on it has been in by a number of we not an effect on when InsP6 levels were elevated or not The increase in InsP6 is with an increase in RIP and may provide a mechanism for the protection by an increase in InsP6 are to TNFα-mediated apoptosis Y. P. 1998; Full Text Full Text PDF PubMed Scopus Google likely due to its role recruiting and activating the IκB kinase complex, in the activation of this not to be involved in the other for RIP be RIP is cleaved by caspase 8 TNFα of its receptor, in a and and RIP levels of the of RIP in cells stimulates the FADD and TRADD while expression of the RIP the FADD to TRADD Y. Y. 1999; PubMed Scopus Google Scholar). RIP can for with to this may result in a of of FADD to TRADD and thus caspase results are with the work by Sun et al. (12Sun Y. Mochizuki Y. Majerus P.W. J. Biol. Chem. 2003; 278: 43645-43653Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) that suggested that the protection from TNFα-mediated apoptosis was due to an inhibition of the of FADD to It is not InsP6 RIP to determine the effect of InsP6 on the of RIP show that cells deficient in InsP6 RIP more when with at RIP levels to of the of RIP in the 2-kinase RNAi lines, while they at of the levels in the luciferase lines not The role of InsP6 is in RIP is a protein with a relatively it may more of to a of for of InsP6 may its rate by the or its for by We Chang for and this