g protein-independent activation of phospholipase c-Δ1 (plc-Δ1) in intestinal smooth muscle is...

April 2000

628

1,25-DIHYDROXYVITAMIN D3 STIMULATES THE RAS, MEK,ERK SIGNALING PATHWAY IN RAT COLONOCYTES.Sharad Khare, Marc Bissonnette, Hemant Roy, William Suh, Michael D.Sitrin, Thomas A. Brasitus, Univ of Chicago, Chicago, IL.

While many actions of I, 25-dihydroxyvitamin D3 (l,25(OHlzD3) involvealterations in gene transcription initiated by the vitamin D nuclear receptor(VDR), this secosteroid induces a number of events too rapid to involvegene transcription. Our laboratory, for example, has previously reportedthat l,25(OHlzD3 rapidly stimulated (sec-min) polyphosphoinositide hy­drolysis, increased intracellular calcium and activated PKC-ain rat colono­cytes. We have also previously demonstrated that l,25(OH)2D3stimulatedproliferation in normal rat colonic epithelium, in contrast to the growthinhibitory effects of this secosteroid in colon cancer-derived cells. Sincethe Ras-MEK-ERK pathway is frequently involved in cellular prolifera­tion, we examined the ability of 1,25(OH)2D3to activate this pathway in ratcolonocytes. Methods: Isolated rat colonocytes were stimulated with1,25(OHlzD3 or the phorbol ester, TPA, for the indicated times andconcentrations. Activated Ras was isolated using agarose beads coupled toGlutathione-S-Transferase fused to the domain of c-Raf that binds acti­vated (GTP-bound) Ras. Activated Ras was detected by Western blottingwith anti Ras antibodies. Activated MEK and MAPK (ERK-I, 2) weredetected by Western blotting with anti-phospho MEK and anti-phosphoMAPK antibodies, respectively. Alterations in the kinase activities ofERK-1 and ERK-2 were confirmed using immune complex kinase assaysin the presence of [y 2p] ATP and myelin basic protein as substrate.Results: 1,25(OHlzD3 (100 nM), but not TPA, rapidly, but transiently (\-5min) activated Ras and MEK-I (2-fold). In contrast, both 1,25(OH)zD3andTPA (200 nM) stimulated ERK-I activity 3-fold and 5-fold, respectively.The PKC inhibitor, calphostin C, abolished the activation of ERK-I byTPA, 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, whileTPA stimulates ERK-I by a RAS-MEK independent pathway. ERK-Iactivation by this secosteroid, moreover, involves PKC-dependent andindependent mechanisms. The mitogenic effects of l,25(OHhD3in normalrat colon are likely mediated, at least in part, by this pathway which isknown to drive proliferation in many other cell types.

629

COUPLING OF THE IGF-I RECEPTOR TYROSINE KINASE TOGIl IN HUMAN INTESTINAL MUSCLE: GBr-DEPENDENT MAPKINASE 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 ofcultured human intestinal muscle by activating distinct MAP kinase- and PI3-kinase-dependent signaling pathways. In these cells cAMP inhibitsgrowth. The aim of this study was to determine whether the IGF-I receptortyrosine kinase activates a heterotrimeric G-protein and to identify the roleof each G-protein subunit in IGF-I-induced growth. Human intestinalsmooth muscle cells isolated from the circular layer were cultured inDMEM+10% serum and used at confluence in first passage. Cells wereincubated in DMEM for 24h and then with 100 nM IGF-I. Growth wasmeasured by [3H]thymidine incorporation. MAP kinase and PI 3-kinaseactivity were measured by in vitro kinase assay. Activation of G-proteinswas measured from the binding of e2S]GTPyS-G~ complexes to specificGa-antibodies. cAMP was measured by RIA. Pertussis toxin (PTx, 200ng/ml) was used to block G, activation. Forskolin (10 11M) was used toincrease cAMP levels. Results: IGF-I caused an increase in [3H]thymidineincorporation (l89:!::9% above basal) that was inhibited in the presence ofPTx (33:!::3% inhibition) or forskolin (62:!::3% inhibition). The effect ofPTx 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 increasedthe binding of [35S]GTPyS-G

a complexes to Gai2 antibody (252:!:: 17%above basal), but not to other G-protein subunit antibodies(G~;l' Gai3, orGqJll)' Binding to Gai2 antibody was inhibited 78:!::4% by the IGF-Ireceptor tyrosine kinase inhibitor, AGI024 (100 roM). The IGF-I-inducedincrease in MAP kinase activity could be also inhibited by imrnunoneu­tralization of Gfl.., subunits(97:!::9%inhibition) derived from Gi2 but not byimmunoneutrahzation 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 whichresults in concurrent IGF-I-induced: (i) G13..,-dependent MAP kinase acti­vation and growth; and (ii) Gai2-dependent inhibition of cAMP productionand disinhibition from cAMP-mediated growth suppression.

AGAA89

630

CERAMIDE REGULATES THE TRANSCRIPTION OF INTER­LEUKIN-8 IN HUMAN PANCREATIC CANCER CELL LINES.Atsushi Masamune, Yoshitaka Sakai, Tooru Shimosegawa, Tohoku UnivSch of Medicine, Sendai, Japan.

(Background) Ceramide signal transduction pathway is known to mediatethe action of various cytokines. Interleukin (IL)-8 is a major inflammatorymediator implicated in several pathological states such as neutrophil­mediated lung injury, tumor growth and metastasis of human liver andpancreatic cancer cells. Here we examined the role of ceramide in theregulation 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-immunosorbentassay. IL-8 mRNA level was determined by Northern analysis. SpecificDNA binding activity and transcriptional activity dependent of transcrip­tion factors were examined by electrophoretic mobility shift assay andluciferase 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-2cells and subsequent experiments were performed using this cell line. Thiseffect 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 sitecompletely and that of AP-1 site partially abrogated the induction ofluciferase activity. C2-ceramide increased NF-kB-specific DNA bindingactivity. Supershift assay revealed that the increased binding activity con­sisted of p50/p65 heterodimer. In addition, ceramide-dependent inductionof IL-8 expression was partially blocked by a specific p38 mitogen­activated protein kinase (MAPK) inhibitor SB203580, and by PD98059, aspecific inhibitor of MAP kinase kinase activation (which prevents activa­tion of extracellular signal-regulated kinase 1/2 (ERK 1/2)). C2-ceramidealso induced IL-8 expression in several other cell lines such as fibroblasts,human vascular endothelial cells, suggesting that ceramide' s action was notcell-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 p38MAPK, in IL-8 production in human pancreatic cancer cell lines.

631

G PROTEIN-INDEPENDENT ACTIVATION OF PHOSPHO­LIPASE C-.11 (PLC-.11) IN INTESTINAL SMOOTH MUSCLE ISMEDIATED BY CAPACITATIVE CAH INFLUX.Karnam S. Murthy, Gabriel M. Makhlouf, Med Coil of Virginia, Rich­mond, VA.

We have previously shown that several phosphoinositide-specific PLCisozymes, including PLC-J31, PLC-J32, PLC-J33, PLC-J34, PLC-y1, andPLC-81, are expressed in intestinal smooth muscle: PLC-J31 and PLC-J33are activated by G protein-coupled receptors, and PLC-y1 by receptortyrosine kinases. The mechanism of activation of PLC-8 has not beenestablished. The presence of IP3 binding sites in the pleckstrin homologydomain of PLC-81, and Ca2+ binding sites in the catalytic and non­catalytic C2 domains, suggests regulation of this isozyme by Ca2+ and IP3.Aim: The aim of the present study was to characterize the regulation ofPLC-81 by Ca2+, IP3 and PKC. Methods: PLC activity (total inositolphosphate formation) was measured in dispersed rabbit intestinal smoothmuscle cells labeled with 3H-inositol. Measurements were made in perme­abilized muscle cells, and in intact muscle cells in which sarcoplasmicCaz+ stores were depleted by 30-min treatment with the sarcoplsmicCaz+/ATPase inhibitor, thapsigargin. Results: In permeabilized musclecells, changing the Ca2+ concentration in the physiological range (0.2 JLMto I fLM) stimulated PLC activity (maximal stimulation 147:!:: 14%). Caz+_

stimulated PLC activity was abolished by antibody to PLC-81, but not byantibodies to PLC-J3I, PLC-J32, PLC-J33, PLC-J34, and PLC-y1, and wasnot affected by blockakde of G protein activation with GDPJ3S. Additionof IP3 (1 fLM) to permeabilized muscle cells or activation of PKC in intactcells by phorbol l2-myristate, 13-acetate (1 fLM) inhibited Ca2+-inducedPLC activity by 59:!::7% and 68:!::7%, respectively. In intact muscle cellsincubated in 0 Ca2+, agonist-independent Ca2+ release induced by thap­sigargin did not activate PLC. Restoration of extracellular Ca2+ followin\Sdepletion of Caz+ stores with thapsigargin, induced capacitative Cazinflux and stimulated PLC activity by 141:!::21 %. Blockade of capacitativeCaz+ influx with SKF-96365 (1 fLM), a non-selective inhibitor of store­operated (SOCCs) and voltage-operated (VOCCs) Ca2+ channels, abol­ished activation of PLC. Conclusion: This study identifies a novel para­digm which links capacitative Caz+ influx to activation of PLC-81, andagonist-induced generation of IP3 and activation of PKC to inhibition ofPLC-81. Activation ofPLC-81 during the phase of capacitative Ca2+ influxis masked by sustained activation of PKC.