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On the relationship between reactive oxygen species, NF-κB activation, and programmed cell death
Abstracts
P5 October 2: Signal Transduction
I 559
and Gene Regulation
Al I20
Al 23 ACTIVATION OF MULTIPLE PROTEIN KINASES, STAT FACTORS AND A H7-SENSITIVE PATHWAY BY GP130STIMULATION T. Hirano, K. Nakajima, T. Matsuda, Y. Fujitani, H. Kojima, Y Yamanaka, M. Takahashi and T. Fukada. Biomed. Res. Ctr., Osaka Univ. Med. Sch., Osaka, Japan IL-6 receptor consists of two molecules: 80 kDa IL-6 receptor (IL-6Ra) and gp130, a common signal transducer for the IL-6 family cytokine. We showed that the activation of gp130 induced a rapid tyrosinephosphorylation of multiple proteins including gp130, Jak family tyrosine kinase which associates with gp130, Stat family factors and a novel Stat-associated 72 kDa tyrosine kinase (p72sak) in hematopeietic lineage cell lines. We further showed that common signals activate the two IL-6 response elements in the promoters of the junB and the acute phase reactant genes through Stat family factors and the Raindependent H7-sensitive pathway is essential for Stat family proteins to be transcriptionally active. We purified the IL-6. induced DNA binding protein with MW of 89kDa from IL-6. stimulated rat liver. Furthermore, we cloned a Stat family protein
which was found to be identical with Stat31ARPF. against Stat3/APRF reacted with the purified activates StatB/APRF or related molecules.
The antibody
p89, showing that IL-6
ON THE RELATIONSHIP BETWEEN REACTIVE OXYGEN SPECIES, NF-KB ACTIVATION, AND PROGRAMMED CELL DEATH Hugo Albrecht and C. Victor Jongeneel Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland Nuclear translocation of the transcription factor NF-KB, which controls the expression of many components of the inflammatory response, has been shown in several cell types to be dependent on the Presence of reactive oxygen species (ROI), particularly OH* and H202. In cells of the myelofd lineage, which produce ROI in their lysosomal compartment through the cytochrome oxidase pathway, NF-aB activation by endotoxin was completely insensffive to ROI scavengers but could be inhibited by phospholipase inhibitors. In T lymphocytes however, activation by phorbol esters and Ca*+ or bv TNF was hiahlv sensftive to ROI scavenaers as well as phospholipase inf&ftors; surprisingly! T cells contained high-amounts of H202 in their cytoplasm even before actwation. In fibroblasts, only the late phase of TNF-induced NF-r;B activation, which seems to coincide with ROI production, was sensitive to the scavengers. Overexpression of the Bcl-2 proto-oncogene protected fibroblasts from H202-induced NF-KB activation and TNF-induced cell death, but not from TNF-induced NF-KB activation. From these experiments and others, we conclude that the presence of ROI may be necessary for the activation of NF-rB in cells that produce ROI through the respiratory chain in preparation for apcptotic death, but is not necessary for this activation under all circumstances.
A121
Al24 Modulated bindine of a 38kDa motein to TNFd mRNA S’UTR.
TRANSCRIPTIONAL CONTROL OF LT-P GENE IN JURKAT CELLS. D.V.Kuprash’t2, D.K.Pokholok’,*, O.A.Osipovich3, M.B.Alimzhanov’, S.V.Kozlovl, R.L.Turetskaya1,2 and S.A.Nedospa~v’,~ ‘Engelhardt Institute of Mol. Biology, Rus.Acad.Sci., Moscow, Russia; 2BCDP, PRI/DynCorp., and 3Lab. of Mol. Immunoregulation, BRMP, NCI-FCRDC, Frederick, MD 21702, USA
PMA res onse in transfections was’lost after deletion of the reeion i-1 r 0 to -80) relative to the transcriotion initiation site. Containing NF-kB and Ets binding sites. Furthermore, point mut: agenesis of the three potential Ets sites abolished PMA inducibility completely. However, the only strongly PMA-inducible binding was that of an ECR-l-related protein to the (-55 to -47) region, i.e. outside the putative PMA-res onsive element. Experiments are underway to determine whet Rer an interplay between different nuclear proteins takes place in this system.
Al25
Al22 TRANSCRIPTIONAL REGULATION OF INTRACELLULAR INTERLEUKIN-1 RECEPTOR ANTAGONIST (icll-lral. John K. Jenkins, Michael F. Smith, Jr. and William P. Arend, University of Colorado Health Sciences Center, Denver, Colorado, 80262. Downregulation of IL-1 ra gene expression is one possible explanation for
inadequate amounts of IL-lra to inhibit the biologic effects of IL-1 in rheumatoid synovial tissue. The recently described epithelial variant of IL1ra (iclL-1 ra) may also be produced by inflammatory cells. To better understand the regulation of iclL-1 ra production we cloned 4555 bp of sequence 5’ of the icll-lra transcriptional initiation start site into a
luciferase expression vector lplC4555Lucl. Deletional mutants were constructed and transient transfection studies performed. Endogenous iclL1 ra mRNA IRT-PCRI and protein (Western
Human keratinocyte
analysis) were also examined.
cell lines IA431 and HaCat) and other epithelial cells
(HT.29 colon carcinoma) express endogenous icll-lra mRNA and protein. In these cells plC4555Luc was also constitutively active. Human peripheral blood monocytes and human and murine monocyteimacrophage cell lines show no constitutive expression of iclL-1 ra. These cells do produce iclL-1 ra mRNA and protein with longer periods of LPS stimulation than that needed
to induce secreted
IL-1 ra fslL-1 ra), suggesting
regulation by an autocrine
factor. The muriw macrophage cell line RAW264.7 demonstrates the same pattern of plC4555Luc activity in response to LPS. Studies with deletional mutants of the icll-lra promoter indicate that different elements are
responsible
for the cell type-specificity
icll-lra gene expression. This work wes supported
of constitutive
by NIH ROlAR39950.
and LPS-inducible
THE ROLE OF MEMBERS OF THE JAK KINASE G-CSF SIGNALLING IN MYELOID CELL LINES.
FAMILY
IN
J.E. Layton, S.E. Nicholson, A. Hqur, A. Ziemieckil and A. Wilks. Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Australia and 1Institute for Clinical and Experimental Cancer Research, Beme, Switzerland. JAKl is associated with the G-CSF receptor (G-CSF-R) in the human AML-193 cell line and in CHO-Kl cells transfected with the G-CSF-R. JAKl and G-CSF-R are tyrosine phosphorylated in response to G-CSF stimulation and JAKl shows increased autophosphorylation in an in vitro kinase assay (Nicholson et al. 1994, Proc. Natl. Acad. Sci. USA, 91: 2985). Since other members of the JAK family have been implicated in cytokine signalling, we have examined the involvement of JAK2 and Tyk2 in the G-CSF response. In AML-193 cells, JAK2 and Tyk2 were both tyrosine phosphorylated in response to G-CSF. Similarly, in BA/F3 cells expressing the murine G-CSF-R, all three JAK kinases were phosphorylated. However, in FDC-PI cells expressing the human G-CSF-R, no JAK phosphorylation was detected, even though JAKl and JAK2 (but not Tyk2) were easily detectable. These results suggest that G-CSF-R can use at least three JAK kinases in signalling, but there is variation between cell lines.
P5 October 2: Signal Transduction
I 559
and Gene Regulation
Al I20
Al 23 ACTIVATION OF MULTIPLE PROTEIN KINASES, STAT FACTORS AND A H7-SENSITIVE PATHWAY BY GP130STIMULATION T. Hirano, K. Nakajima, T. Matsuda, Y. Fujitani, H. Kojima, Y Yamanaka, M. Takahashi and T. Fukada. Biomed. Res. Ctr., Osaka Univ. Med. Sch., Osaka, Japan IL-6 receptor consists of two molecules: 80 kDa IL-6 receptor (IL-6Ra) and gp130, a common signal transducer for the IL-6 family cytokine. We showed that the activation of gp130 induced a rapid tyrosinephosphorylation of multiple proteins including gp130, Jak family tyrosine kinase which associates with gp130, Stat family factors and a novel Stat-associated 72 kDa tyrosine kinase (p72sak) in hematopeietic lineage cell lines. We further showed that common signals activate the two IL-6 response elements in the promoters of the junB and the acute phase reactant genes through Stat family factors and the Raindependent H7-sensitive pathway is essential for Stat family proteins to be transcriptionally active. We purified the IL-6. induced DNA binding protein with MW of 89kDa from IL-6. stimulated rat liver. Furthermore, we cloned a Stat family protein
which was found to be identical with Stat31ARPF. against Stat3/APRF reacted with the purified activates StatB/APRF or related molecules.
The antibody
p89, showing that IL-6
ON THE RELATIONSHIP BETWEEN REACTIVE OXYGEN SPECIES, NF-KB ACTIVATION, AND PROGRAMMED CELL DEATH Hugo Albrecht and C. Victor Jongeneel Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland Nuclear translocation of the transcription factor NF-KB, which controls the expression of many components of the inflammatory response, has been shown in several cell types to be dependent on the Presence of reactive oxygen species (ROI), particularly OH* and H202. In cells of the myelofd lineage, which produce ROI in their lysosomal compartment through the cytochrome oxidase pathway, NF-aB activation by endotoxin was completely insensffive to ROI scavengers but could be inhibited by phospholipase inhibitors. In T lymphocytes however, activation by phorbol esters and Ca*+ or bv TNF was hiahlv sensftive to ROI scavenaers as well as phospholipase inf&ftors; surprisingly! T cells contained high-amounts of H202 in their cytoplasm even before actwation. In fibroblasts, only the late phase of TNF-induced NF-r;B activation, which seems to coincide with ROI production, was sensitive to the scavengers. Overexpression of the Bcl-2 proto-oncogene protected fibroblasts from H202-induced NF-KB activation and TNF-induced cell death, but not from TNF-induced NF-KB activation. From these experiments and others, we conclude that the presence of ROI may be necessary for the activation of NF-rB in cells that produce ROI through the respiratory chain in preparation for apcptotic death, but is not necessary for this activation under all circumstances.
A121
Al24 Modulated bindine of a 38kDa motein to TNFd mRNA S’UTR.
TRANSCRIPTIONAL CONTROL OF LT-P GENE IN JURKAT CELLS. D.V.Kuprash’t2, D.K.Pokholok’,*, O.A.Osipovich3, M.B.Alimzhanov’, S.V.Kozlovl, R.L.Turetskaya1,2 and S.A.Nedospa~v’,~ ‘Engelhardt Institute of Mol. Biology, Rus.Acad.Sci., Moscow, Russia; 2BCDP, PRI/DynCorp., and 3Lab. of Mol. Immunoregulation, BRMP, NCI-FCRDC, Frederick, MD 21702, USA
PMA res onse in transfections was’lost after deletion of the reeion i-1 r 0 to -80) relative to the transcriotion initiation site. Containing NF-kB and Ets binding sites. Furthermore, point mut: agenesis of the three potential Ets sites abolished PMA inducibility completely. However, the only strongly PMA-inducible binding was that of an ECR-l-related protein to the (-55 to -47) region, i.e. outside the putative PMA-res onsive element. Experiments are underway to determine whet Rer an interplay between different nuclear proteins takes place in this system.
Al25
Al22 TRANSCRIPTIONAL REGULATION OF INTRACELLULAR INTERLEUKIN-1 RECEPTOR ANTAGONIST (icll-lral. John K. Jenkins, Michael F. Smith, Jr. and William P. Arend, University of Colorado Health Sciences Center, Denver, Colorado, 80262. Downregulation of IL-1 ra gene expression is one possible explanation for
inadequate amounts of IL-lra to inhibit the biologic effects of IL-1 in rheumatoid synovial tissue. The recently described epithelial variant of IL1ra (iclL-1 ra) may also be produced by inflammatory cells. To better understand the regulation of iclL-1 ra production we cloned 4555 bp of sequence 5’ of the icll-lra transcriptional initiation start site into a
luciferase expression vector lplC4555Lucl. Deletional mutants were constructed and transient transfection studies performed. Endogenous iclL1 ra mRNA IRT-PCRI and protein (Western
Human keratinocyte
analysis) were also examined.
cell lines IA431 and HaCat) and other epithelial cells
(HT.29 colon carcinoma) express endogenous icll-lra mRNA and protein. In these cells plC4555Luc was also constitutively active. Human peripheral blood monocytes and human and murine monocyteimacrophage cell lines show no constitutive expression of iclL-1 ra. These cells do produce iclL-1 ra mRNA and protein with longer periods of LPS stimulation than that needed
to induce secreted
IL-1 ra fslL-1 ra), suggesting
regulation by an autocrine
factor. The muriw macrophage cell line RAW264.7 demonstrates the same pattern of plC4555Luc activity in response to LPS. Studies with deletional mutants of the icll-lra promoter indicate that different elements are
responsible
for the cell type-specificity
icll-lra gene expression. This work wes supported
of constitutive
by NIH ROlAR39950.
and LPS-inducible
THE ROLE OF MEMBERS OF THE JAK KINASE G-CSF SIGNALLING IN MYELOID CELL LINES.
FAMILY
IN
J.E. Layton, S.E. Nicholson, A. Hqur, A. Ziemieckil and A. Wilks. Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Australia and 1Institute for Clinical and Experimental Cancer Research, Beme, Switzerland. JAKl is associated with the G-CSF receptor (G-CSF-R) in the human AML-193 cell line and in CHO-Kl cells transfected with the G-CSF-R. JAKl and G-CSF-R are tyrosine phosphorylated in response to G-CSF stimulation and JAKl shows increased autophosphorylation in an in vitro kinase assay (Nicholson et al. 1994, Proc. Natl. Acad. Sci. USA, 91: 2985). Since other members of the JAK family have been implicated in cytokine signalling, we have examined the involvement of JAK2 and Tyk2 in the G-CSF response. In AML-193 cells, JAK2 and Tyk2 were both tyrosine phosphorylated in response to G-CSF. Similarly, in BA/F3 cells expressing the murine G-CSF-R, all three JAK kinases were phosphorylated. However, in FDC-PI cells expressing the human G-CSF-R, no JAK phosphorylation was detected, even though JAKl and JAK2 (but not Tyk2) were easily detectable. These results suggest that G-CSF-R can use at least three JAK kinases in signalling, but there is variation between cell lines.