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Ceramide regulates the transcription of interleukin-8 in human pancreatic cancer cell lines
April 2000
AGAA89
628
630
1,25-DIHYDROXYVITAMIN D3 STIMULATES THE RAS, MEK, ERK SIGNALING PATHWAY IN RAT COLONOCYTES.
CERAMIDE REGULATES THE TRANSCRIPTION OF INTERLEUKIN-8 IN HUMAN PANCREATIC CANCER CELL LINES.
Sharad Khare, Marc Bissonnette, Hemant Roy, William Suh, Michael D. Sitrin, Thomas A. Brasitus, Univ of Chicago, Chicago, IL.
Atsushi Masamune, Yoshitaka Sakai, Tooru Shimosegawa, Tohoku Univ Sch of Medicine, Sendai, Japan.
While many actions of I, 25-dihydroxyvitamin D3 (l,25(OHlzD 3) involve alterations in gene transcription initiated by the vitamin D nuclear receptor (VDR), this secosteroid induces a number of events too rapid to involve gene transcription. Our laboratory, for example, has previously reported that l,25(OHlzD 3 rapidly stimulated (sec-min) polyphosphoinositide hydrolysis, increased intracellular calcium and activated PKC-ain rat colonocytes. We have also previously demonstrated that l,25(OH)2D3stimulated proliferation in normal rat colonic epithelium, in contrast to the growth inhibitory effects of this secosteroid in colon cancer-derived cells. Since the Ras-MEK-ERK pathway is frequently involved in cellular proliferation, we examined the ability of 1,25(OH)2D3to activate this pathway in rat colonocytes. Methods: Isolated rat colonocytes were stimulated with 1,25(OHlzD3 or the phorbol ester, TPA, for the indicated times and concentrations. Activated Ras was isolated using agarose beads coupled to Glutathione-S-Transferase fused to the domain of c-Raf that binds activated (GTP-bound) Ras. Activated Ras was detected by Western blotting with anti Ras antibodies. Activated MEK and MAPK (ERK-I, 2) were detected by Western blotting with anti-phospho MEK and anti-phospho MAPK antibodies, respectively. Alterations in the kinase activities of ERK-1 and ERK-2 were confirmed using immune complex kinase assays in the presence of [y 2 p] ATP and myelin basic protein as substrate. Results: 1,25(OHlzD3 (100 nM), but not TPA, rapidly, but transiently (\-5 min) activated Ras and MEK-I (2-fold). In contrast, both 1,25(OH)zD3and TPA (200 nM) stimulated ERK-I activity 3-fold and 5-fold, respectively. The PKC inhibitor, calphostin C, abolished the activation of ERK-I by TPA, but only partially inhibited that caused by l,25(OH)2D3' In summary, 1,25(OHlzD3 activates Ras, MEK-I and ERK-I in rat colonocytes, while TPA stimulates ERK-I by a RAS-MEK independent pathway. ERK-I activation by this secosteroid, moreover, involves PKC-dependent and independent mechanisms. The mitogenic effects of l,25(OHhD 3 in normal rat colon are likely mediated, at least in part, by this pathway which is known to drive proliferation in many other cell types.
(Background) Ceramide signal transduction pathway is known to mediate the action of various cytokines. Interleukin (IL)-8 is a major inflammatory mediator implicated in several pathological states such as neutrophilmediated lung injury, tumor growth and metastasis of human liver and pancreatic cancer cells. Here we examined the role of ceramide in the regulation of IL-8 production in human pancreatic cancer cells. (Methods) Three human pancreatic cancer cell lines (MiaPaca-2, Pane-L, BxPC-3) were used. IL-8 production was examined by enzyme-immunosorbent assay. IL-8 mRNA level was determined by Northern analysis. Specific DNA binding activity and transcriptional activity dependent of transcription factors were examined by electrophoretic mobility shift assay and luciferase assay, respectively. (Results) A cell-permeable ceramide analog (C2-ceramide) increased IL-8 expression with comparable mRNA induction. Sphingosine also increased IL-8 secretion, but to a lesser degree. Neither C2-dihydroceramide, an inactive analogue, nor sphingosine-1phosphate were effective. The effect was most pronounced in MiaPaca-2 cells and subsequent experiments were performed using this cell line. This effect was dependent on nuclear factor kB (NF-kB) and, to a lesser extent, activator protein-I (AP-1), because mutation of the NF-kB binding site completely and that of AP-1 site partially abrogated the induction of luciferase activity. C2-ceramide increased NF-kB-specific DNA binding activity. Supershift assay revealed that the increased binding activity consisted of p50/p65 heterodimer. In addition, cerami de-dependent induction of IL-8 expression was partially blocked by a specific p38 mitogenactivated protein kinase (MAPK) inhibitor SB203580, and by PD98059, a specific inhibitor of MAP kinase kinase activation (which prevents activation of extracellular signal-regulated kinase 1/2 (ERK 1/2)). C2-ceramide also induced IL-8 expression in several other cell lines such as fibroblasts, human vascular endothelial cells, suggesting that ceramide' s action was not cell-type specific. (Conclusion) Our results suggest a novel role of ceramide, at least in part through NF-kB and AP-I as well as ERK 1/2 and p38 MAPK, in IL-8 production in human pancreatic cancer cell lines.
629 COUPLING OF THE IGF-I RECEPTOR TYROSINE KINASE TO GIl IN HUMAN INTESTINAL MUSCLE: GBr-DEPENDENT MAP KINASE ACTIVATION AND GROWTH. John F. Kuemmerle, Kamam S. Murthy, Med Col of VA (VCU), Richmond, VA. Endogenous Insulin-like growth factor-I (IGF-I) stimulates growth of cultured human intestinal muscle by activating distinct MAP kinase- and PI 3-kinase-dependent signaling pathways. In these cells cAMP inhibits growth. The aim of this study was to determine whether the IGF-I receptor tyrosine kinase activates a heterotrimeric G-protein and to identify the role of each G-protein subunit in IGF-I-induced growth. Human intestinal smooth muscle cells isolated from the circular layer were cultured in DMEM + 10% serum and used at confluence in first passage. Cells were incubated in DMEM for 24h and then with 100 nM IGF-I. Growth was measured by [3H]thymidine incorporation. MAP kinase and PI 3-kinase activity were measured by in vitro kinase assay. Activation of G-proteins was measured from the binding of e2S]GTPyS-G~ complexes to specific Ga-antibodies. cAMP was measured by RIA. Pertussis toxin (PTx, 200 ng/ml) was used to block G, activation. Forskolin (10 11M) was used to increase cAMP levels. Results: IGF-I caused an increase in [3H]thymidine incorporation (l89:!::9% above basal) that was inhibited in the presence of PTx (33:!::3% inhibition) or forskolin (62:!::3% inhibition). The effect of PTx was augmented by a PI 3-kinase inhibitor, LY294002 (Il: 54:!:: 1%), but not by a MAP kinase inhibitor, PD98059. IGF-I caused a timedependent increase in MAP kinase activity (10 min: 5.2:!:: 1.2; 30 min: 2.8:!::0.5 pmol Pi/min/mg protein above basal) that was inhibited by PTx (10 min: 48:':16% and 30 min: 100:!::5% inhibition). In contrast, IGF-Iinduced PI 3-kinase activation was not affected by PTx. IGF-I increased the binding of [35S]GTPyS-Ga complexes to Gai2 antibody (252:!:: 17% above basal), but not to other G-protein subunit antibodies(G~;l' G ai3, or GqJll)' Binding to Gai2 antibody was inhibited 78:!::4% by the IGF-I receptor tyrosine kinase inhibitor, AGI024 (100 roM). The IGF-I-induced increase in MAP kinase activity could be also inhibited by imrnunoneutralization of Gfl.., subunits(97:!::9%inhibition) derived from Gi2 but not by immunoneutrahzation of Ga i2 subunits. IGF-I inhibited basal cAMP levels(30:':4% inhibition) and forskolin-stimulated cAMP production (89:!:: I% inhibition). This effect was reversed in the presence of PTx. Conclusion: The IGF-I rececptor tyrosine kinase is coupled to Gil which results in concurrent IGF-I-induced: (i) G13..,-dependent MAP kinase activation and growth; and (ii) Gai2-dependent inhibition of cAMP production and disinhibition from cAMP-mediated growth suppression.
631
G PROTEIN-INDEPENDENT ACTIVATION OF PHOSPHOLIPASE C-.11 (PLC-.11) IN INTESTINAL SMOOTH MUSCLE IS MEDIATED BY CAPACITATIVE CAH INFLUX. Karnam S. Murthy, Gabriel M. Makhlouf, Med Coil of Virginia, Richmond, VA. We have previously shown that several phosphoinositide-specific PLC isozymes, including PLC-J31, PLC-J32, PLC-J33, PLC-J34, PLC-y1, and PLC-81, are expressed in intestinal smooth muscle: PLC-J31 and PLC-J33 are activated by G protein-coupled receptors, and PLC-y1 by receptor tyrosine kinases. The mechanism of activation of PLC-8 has not been established. The presence of IP3 binding sites in the pleckstrin homology domain of PLC-81, and Ca 2+ binding sites in the catalytic and noncatalytic C2 domains, suggests regulation of this isozyme by Ca 2+ and IP3. Aim: The aim of the present study was to characterize the regulation of PLC-81 by Ca 2+, IP3 and PKC. Methods: PLC activity (total inositol phosphate formation) was measured in dispersed rabbit intestinal smooth muscle cells labeled with 3H-inositol. Measurements were made in permeabilized muscle cells, and in intact muscle cells in which sarcoplasmic Ca z+ stores were depleted by 30-min treatment with the sarcoplsmic Caz+/ATPase inhibitor, thapsigargin. Results: In permeabilized muscle cells, changing the Ca2+ concentration in the physiological range (0.2 JLM to I fLM) stimulated PLC activity (maximal stimulation 147:!:: 14%). Ca z+_ stimulated PLC activity was abolished by antibody to PLC-81, but not by antibodies to PLC-J3I, PLC-J32, PLC-J33, PLC-J34, and PLC-y1, and was not affected by blockakde of G protein activation with GDPJ3S. Addition of IP 3 (1 fLM) to permeabilized muscle cells or activation of PKC in intact cells by phorbol l2-myristate, 13-acetate (1 fLM) inhibited Ca 2+ -induced PLC activity by 59:!::7% and 68:!::7%, respectively. In intact muscle cells incubated in 0 Ca2+, agonist-independent Ca 2+ release induced by thapsigargin did not activate PLC. Restoration of extracellular Ca 2+ followin\S depletion of Ca z+ stores with thapsigargin, induced capacitative Caz influx and stimulated PLC activity by 141:!::21 %. Blockade of capacitative Ca z+ influx with SKF-96365 (1 fLM), a non-selective inhibitor of storeoperated (SOCCs) and voltage-operated (VOCCs) Ca 2+ channels, abolished activation of PLC. Conclusion: This study identifies a novel paradigm which links capacitative Ca z+ influx to activation of PLC-81, and agonist-induced generation of IP3 and activation of PKC to inhibition of PLC-81. Activation ofPLC-81 during the phase of capacitative Ca 2+ influx is masked by sustained activation of PKC.
AGAA89
628
630
1,25-DIHYDROXYVITAMIN D3 STIMULATES THE RAS, MEK, ERK SIGNALING PATHWAY IN RAT COLONOCYTES.
CERAMIDE REGULATES THE TRANSCRIPTION OF INTERLEUKIN-8 IN HUMAN PANCREATIC CANCER CELL LINES.
Sharad Khare, Marc Bissonnette, Hemant Roy, William Suh, Michael D. Sitrin, Thomas A. Brasitus, Univ of Chicago, Chicago, IL.
Atsushi Masamune, Yoshitaka Sakai, Tooru Shimosegawa, Tohoku Univ Sch of Medicine, Sendai, Japan.
While many actions of I, 25-dihydroxyvitamin D3 (l,25(OHlzD 3) involve alterations in gene transcription initiated by the vitamin D nuclear receptor (VDR), this secosteroid induces a number of events too rapid to involve gene transcription. Our laboratory, for example, has previously reported that l,25(OHlzD 3 rapidly stimulated (sec-min) polyphosphoinositide hydrolysis, increased intracellular calcium and activated PKC-ain rat colonocytes. We have also previously demonstrated that l,25(OH)2D3stimulated proliferation in normal rat colonic epithelium, in contrast to the growth inhibitory effects of this secosteroid in colon cancer-derived cells. Since the Ras-MEK-ERK pathway is frequently involved in cellular proliferation, we examined the ability of 1,25(OH)2D3to activate this pathway in rat colonocytes. Methods: Isolated rat colonocytes were stimulated with 1,25(OHlzD3 or the phorbol ester, TPA, for the indicated times and concentrations. Activated Ras was isolated using agarose beads coupled to Glutathione-S-Transferase fused to the domain of c-Raf that binds activated (GTP-bound) Ras. Activated Ras was detected by Western blotting with anti Ras antibodies. Activated MEK and MAPK (ERK-I, 2) were detected by Western blotting with anti-phospho MEK and anti-phospho MAPK antibodies, respectively. Alterations in the kinase activities of ERK-1 and ERK-2 were confirmed using immune complex kinase assays in the presence of [y 2 p] ATP and myelin basic protein as substrate. Results: 1,25(OHlzD3 (100 nM), but not TPA, rapidly, but transiently (\-5 min) activated Ras and MEK-I (2-fold). In contrast, both 1,25(OH)zD3and TPA (200 nM) stimulated ERK-I activity 3-fold and 5-fold, respectively. The PKC inhibitor, calphostin C, abolished the activation of ERK-I by TPA, but only partially inhibited that caused by l,25(OH)2D3' In summary, 1,25(OHlzD3 activates Ras, MEK-I and ERK-I in rat colonocytes, while TPA stimulates ERK-I by a RAS-MEK independent pathway. ERK-I activation by this secosteroid, moreover, involves PKC-dependent and independent mechanisms. The mitogenic effects of l,25(OHhD 3 in normal rat colon are likely mediated, at least in part, by this pathway which is known to drive proliferation in many other cell types.
(Background) Ceramide signal transduction pathway is known to mediate the action of various cytokines. Interleukin (IL)-8 is a major inflammatory mediator implicated in several pathological states such as neutrophilmediated lung injury, tumor growth and metastasis of human liver and pancreatic cancer cells. Here we examined the role of ceramide in the regulation of IL-8 production in human pancreatic cancer cells. (Methods) Three human pancreatic cancer cell lines (MiaPaca-2, Pane-L, BxPC-3) were used. IL-8 production was examined by enzyme-immunosorbent assay. IL-8 mRNA level was determined by Northern analysis. Specific DNA binding activity and transcriptional activity dependent of transcription factors were examined by electrophoretic mobility shift assay and luciferase assay, respectively. (Results) A cell-permeable ceramide analog (C2-ceramide) increased IL-8 expression with comparable mRNA induction. Sphingosine also increased IL-8 secretion, but to a lesser degree. Neither C2-dihydroceramide, an inactive analogue, nor sphingosine-1phosphate were effective. The effect was most pronounced in MiaPaca-2 cells and subsequent experiments were performed using this cell line. This effect was dependent on nuclear factor kB (NF-kB) and, to a lesser extent, activator protein-I (AP-1), because mutation of the NF-kB binding site completely and that of AP-1 site partially abrogated the induction of luciferase activity. C2-ceramide increased NF-kB-specific DNA binding activity. Supershift assay revealed that the increased binding activity consisted of p50/p65 heterodimer. In addition, cerami de-dependent induction of IL-8 expression was partially blocked by a specific p38 mitogenactivated protein kinase (MAPK) inhibitor SB203580, and by PD98059, a specific inhibitor of MAP kinase kinase activation (which prevents activation of extracellular signal-regulated kinase 1/2 (ERK 1/2)). C2-ceramide also induced IL-8 expression in several other cell lines such as fibroblasts, human vascular endothelial cells, suggesting that ceramide' s action was not cell-type specific. (Conclusion) Our results suggest a novel role of ceramide, at least in part through NF-kB and AP-I as well as ERK 1/2 and p38 MAPK, in IL-8 production in human pancreatic cancer cell lines.
629 COUPLING OF THE IGF-I RECEPTOR TYROSINE KINASE TO GIl IN HUMAN INTESTINAL MUSCLE: GBr-DEPENDENT MAP KINASE ACTIVATION AND GROWTH. John F. Kuemmerle, Kamam S. Murthy, Med Col of VA (VCU), Richmond, VA. Endogenous Insulin-like growth factor-I (IGF-I) stimulates growth of cultured human intestinal muscle by activating distinct MAP kinase- and PI 3-kinase-dependent signaling pathways. In these cells cAMP inhibits growth. The aim of this study was to determine whether the IGF-I receptor tyrosine kinase activates a heterotrimeric G-protein and to identify the role of each G-protein subunit in IGF-I-induced growth. Human intestinal smooth muscle cells isolated from the circular layer were cultured in DMEM + 10% serum and used at confluence in first passage. Cells were incubated in DMEM for 24h and then with 100 nM IGF-I. Growth was measured by [3H]thymidine incorporation. MAP kinase and PI 3-kinase activity were measured by in vitro kinase assay. Activation of G-proteins was measured from the binding of e2S]GTPyS-G~ complexes to specific Ga-antibodies. cAMP was measured by RIA. Pertussis toxin (PTx, 200 ng/ml) was used to block G, activation. Forskolin (10 11M) was used to increase cAMP levels. Results: IGF-I caused an increase in [3H]thymidine incorporation (l89:!::9% above basal) that was inhibited in the presence of PTx (33:!::3% inhibition) or forskolin (62:!::3% inhibition). The effect of PTx was augmented by a PI 3-kinase inhibitor, LY294002 (Il: 54:!:: 1%), but not by a MAP kinase inhibitor, PD98059. IGF-I caused a timedependent increase in MAP kinase activity (10 min: 5.2:!:: 1.2; 30 min: 2.8:!::0.5 pmol Pi/min/mg protein above basal) that was inhibited by PTx (10 min: 48:':16% and 30 min: 100:!::5% inhibition). In contrast, IGF-Iinduced PI 3-kinase activation was not affected by PTx. IGF-I increased the binding of [35S]GTPyS-Ga complexes to Gai2 antibody (252:!:: 17% above basal), but not to other G-protein subunit antibodies(G~;l' G ai3, or GqJll)' Binding to Gai2 antibody was inhibited 78:!::4% by the IGF-I receptor tyrosine kinase inhibitor, AGI024 (100 roM). The IGF-I-induced increase in MAP kinase activity could be also inhibited by imrnunoneutralization of Gfl.., subunits(97:!::9%inhibition) derived from Gi2 but not by immunoneutrahzation of Ga i2 subunits. IGF-I inhibited basal cAMP levels(30:':4% inhibition) and forskolin-stimulated cAMP production (89:!:: I% inhibition). This effect was reversed in the presence of PTx. Conclusion: The IGF-I rececptor tyrosine kinase is coupled to Gil which results in concurrent IGF-I-induced: (i) G13..,-dependent MAP kinase activation and growth; and (ii) Gai2-dependent inhibition of cAMP production and disinhibition from cAMP-mediated growth suppression.
631
G PROTEIN-INDEPENDENT ACTIVATION OF PHOSPHOLIPASE C-.11 (PLC-.11) IN INTESTINAL SMOOTH MUSCLE IS MEDIATED BY CAPACITATIVE CAH INFLUX. Karnam S. Murthy, Gabriel M. Makhlouf, Med Coil of Virginia, Richmond, VA. We have previously shown that several phosphoinositide-specific PLC isozymes, including PLC-J31, PLC-J32, PLC-J33, PLC-J34, PLC-y1, and PLC-81, are expressed in intestinal smooth muscle: PLC-J31 and PLC-J33 are activated by G protein-coupled receptors, and PLC-y1 by receptor tyrosine kinases. The mechanism of activation of PLC-8 has not been established. The presence of IP3 binding sites in the pleckstrin homology domain of PLC-81, and Ca 2+ binding sites in the catalytic and noncatalytic C2 domains, suggests regulation of this isozyme by Ca 2+ and IP3. Aim: The aim of the present study was to characterize the regulation of PLC-81 by Ca 2+, IP3 and PKC. Methods: PLC activity (total inositol phosphate formation) was measured in dispersed rabbit intestinal smooth muscle cells labeled with 3H-inositol. Measurements were made in permeabilized muscle cells, and in intact muscle cells in which sarcoplasmic Ca z+ stores were depleted by 30-min treatment with the sarcoplsmic Caz+/ATPase inhibitor, thapsigargin. Results: In permeabilized muscle cells, changing the Ca2+ concentration in the physiological range (0.2 JLM to I fLM) stimulated PLC activity (maximal stimulation 147:!:: 14%). Ca z+_ stimulated PLC activity was abolished by antibody to PLC-81, but not by antibodies to PLC-J3I, PLC-J32, PLC-J33, PLC-J34, and PLC-y1, and was not affected by blockakde of G protein activation with GDPJ3S. Addition of IP 3 (1 fLM) to permeabilized muscle cells or activation of PKC in intact cells by phorbol l2-myristate, 13-acetate (1 fLM) inhibited Ca 2+ -induced PLC activity by 59:!::7% and 68:!::7%, respectively. In intact muscle cells incubated in 0 Ca2+, agonist-independent Ca 2+ release induced by thapsigargin did not activate PLC. Restoration of extracellular Ca 2+ followin\S depletion of Ca z+ stores with thapsigargin, induced capacitative Caz influx and stimulated PLC activity by 141:!::21 %. Blockade of capacitative Ca z+ influx with SKF-96365 (1 fLM), a non-selective inhibitor of storeoperated (SOCCs) and voltage-operated (VOCCs) Ca 2+ channels, abolished activation of PLC. Conclusion: This study identifies a novel paradigm which links capacitative Ca z+ influx to activation of PLC-81, and agonist-induced generation of IP3 and activation of PKC to inhibition of PLC-81. Activation ofPLC-81 during the phase of capacitative Ca 2+ influx is masked by sustained activation of PKC.