Bc249917202p

Vol. 274, No. 24, Issue of June 11, pp. 17202–17208, 1999 Dexamethasone Alters Arachidonate Release from Human
Epithelial Cells by Induction of p11 Protein Synthesis and
Inhibition of Phospholipase A Activity*
(Received for publication, December 23, 1998, and in revised form, March 26, 1999) Xiang-Lan Yao, Mark J. Cowan, Mark T. Gladwin, Marion M. Lawrence, C. William Angus,
and James H. Shelhamer‡
From the Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892 The effect of the glucocorticosteroid, dexamethasone,
portant role in inflammation (1– 4). The mammalian calcium- on arachidonic acid (AA) release and on protein levels of
dependent PLA s can be grouped into major classes based on p11 and cytosolic phospholipase A (cPLA ) was studied
their molecular mass and cellular distribution, including the in two epithelial cell lines, HeLa cells and BEAS-2B cells.
low molecular mass (10 –14 kDa) secreted forms (sPLA ) and Dexamethasone treatment of HeLa cells and BEAS-2B
the structurally unrelated high molecular mass (85 kDa) cyto- cells increased cellular p11 protein and mRNA levels in
a time- and dose-dependent manner. It had little effect
To date, five different sPLA isozymes have been described in on levels of cPLA protein. In order to determine if in-
mammalian cells. The 14-kDa sPLA enzyme from synovial creased p11 protein expression resulted in increased
fluid and platelets (Group IIA) may be involved in the patho- interaction between p11 and cPLA , anti-cPLA antibod-
genesis of inflammatory reactions (3, 6, 7). The 14-kDa PLA ies were used to immunoprecipitate p11⅐cPLA com-
lacks apparent selectivity for the sn-2 fatty acids of phospho- plexes and Western blots of the immunoprecipitate were
lipids and requires much higher Ca2ϩ concentrations (millimo- used to detect p11. In cells treated with dexamethasone,
lar) than normal intracellular Ca2ϩ levels (nanomolar to mi- more p11 was detected in the anti-cPLA immunopre-
cipitate compared with control cells. Dexamethasone
cromolar) for activity. The 85-kDa high molecular mass cPLA2 treatment of HeLa cells prelabeled with [3H]AA de-
has higher selectivity to hydrolyze phospholipids containing creased the release of [3H]AA under basal conditions
AA esterified in the sn-2 position (1, 3, 5, 8 –11). Its activity is and after stimulation with the calcium ionophore
regulated by phosphorylation, G-protein activation, and phys- A23187 (10؊6 M). In order to determine if altering the p11
iologically relevant concentrations of calcium. Because cPLA2 protein levels in HeLa cells independent of glucocorti-
may play a central role in producing AA and lysophospholipid costeroid treatment could also produce an effect on
for subsequent metabolism to prostaglandins, leukotrienes, hy- [3H]AA release, cells were stably transfected with plas-
droxyeicosatetraenoic acids, and platelet-activating factor, all mids expressing either p11 antisense mRNA or p11
potent lipid mediators of inflammation, the activation of cPLA2 mRNA. Cloned HeLa cells expressing p11 antisense
may play an important role in modulating the airway inflam- mRNA exhibited less cellular p11 protein compared
with control cells and greater [3H]AA release compared
S-100 proteins are a family of proteins first described by with cells transfected with a control vector. Cloned
Moore (12) who initially characterized a group of abundant low HeLa cells stably transfected with a p11 expression vec-
molecular weight (10 –12 kDa) acidic proteins in neural tissue.
tor exhibited increased p11 cellular protein and dimin-
S-100 proteins are a group of Ca2ϩ-binding proteins that are ished [3H]AA release under basal conditions and in re-
expressed in a cell type-dependent fashion. This family in- sponse to A23187. Therefore, dexamethasone alteration
cludes S-100␣, S-100␤, and p11/calpactin light chain (13). p11 of epithelial cell AA release may be due in part to induc-
was described as a member of the S-100 family of EF hand type tion of p11 protein expression.
Ca2ϩ-binding proteins but does not have the ability to bindCa2ϩ ions due to crucial amino acid deletions and substitutionsin the two EF hand loops of the protein (14, 15). p11 binds to Phospholipase A s (PLA s)1 are a group of enzymes that and inhibits the phosphorylation of a 36-kDa protein known as hydrolyze the ester bond of fatty acids from the sn-2 position of p36, also known as annexin II as well as calpactin heavy chain glycerophospholipids. The release of arachidonic acid (AA) from membranes by PLA and its subsequent conversion into leu- Glucocorticoids are effective in the treatment of immune and kotrienes, prostaglandins, and other eicosanoids plays an im- inflammatory disorders affecting the lung and other organs.
One mechanism of glucocorticoid modulation of the inflamma-tory response is inhibition of the release of AA from cellular * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked lipids (18, 19) and inhibition of prostaglandin H synthase-2 “advertisement” in accordance with 18 U.S.C. Section 1734 solely to synthase or cyclooxygenase-2 expression in a number of tissues (20 –24). The rate of eicosanoid synthesis may be regulated by ‡ To whom correspondence should be addressed: Bldg. 10, Rm. 7-D-43, the availability of free AA that can be metabolized into pros- 1 The abbreviations used are: PLA , phospholipase A ; cPLA , cyto- tanoids and leukotrienes via the cyclooxygenase and lipoxyge- solic PLA ; sPLA , secreted PLA ; AA, arachidonic acid; DMEM, Dul- nase pathways. The decreased synthesis of bioactive eico- becco’s modified Eagle’s medium; PBS, phosphate-buffered saline; sanoids may represent an important mechanism of the anti- HBSS, Hanks’ balanced salt solution; RPA, ribonuclease protection inflammatory action of glucocorticoids. Glucocorticoids can assay; bp, base pair(s); GAPDH, glyceradehyde 3-phosphate dehydro- induce annexins which might inhibit sPLA activity in vitro genase; BSA, bovine serum albumin; HPLC, high-pressure liquid (25–30). A recent study has demonstrated that p11 can directly This paper is available on line at http://www.jbc.org
Dexamethasone Alteration of Cellular p11 interact with the COOH-terminal region of 85-kDa cPLA and roform extraction method (Tri-reagent, Molecular Research Inc., Cin- inhibit cPLA enzyme activity (31). Therefore, it was of interest cinnati, OH). The RNA pellet was precipitated with isopropyl alcohol, washed with 70% ethanol, and redissolved in diethyl pyrocarbonate to study whether p11 plays a role in glucocorticoid induced water. To construct the probe for cPLA mRNA, a 306-bp product of changes in cellular arachidonate release.
cPLA cDNA was amplified by polymerase chain reaction using the following sets of sense and antisense primers: 5Ј primer, 5Ј-CTCACA- CCACAGAAAGTTAAAAGAT-3Ј(799 – 823); 3Ј primer, 5Ј-AAATAAGT- Cell Culture—HeLa cells were obtained from the American Type CGGGAGCCATAAA-3Ј (1104 –1084) (Biosynthesis Inc., Lewisville, Culture Collection (Rockville, MD) and grown in DMEM medium with TX). The product for cPLA gene was cloned into the TA cloning vector 10% fetal bovine serum. BEAS-2B cells, a human bronchial epithelial (Invitrogen, San Diego, CA). Orientation of the insert was determined cell line, were a gift from Curtis Harris and John Lechner, National by DNA sequencing. To construct the probe for p11 mRNA, a 320-bp Cancer Institute, Bethesda, MD. BEAS-B cells were grown in LHC-8 product of p11 cDNA was amplified by polymerase chain reaction using medium (Biofluids, Rockville, MD) without hydrocortisone or serum. All the following sets of sense and antisense primers: 5Ј primer, 5Ј-ACCA- experiments were performed when cells were 80% confluent.
CACCAAAATGCCATCTC-3Ј(101–121); 3Ј primer, 5Ј-CTGCTCATTTC- Immunoblot of p11 Protein—HeLa or BEAS-2B cells were grown on TGCCTACTT-3Ј (400 – 419) (Genosys Biotechnologies, Inc., The Wood- 175-cm2 flasks and treated with dexamethasone (Calbiochem) (10Ϫ7, lands, TX). The product for p11 was cloned into the pGEM-T Easy 10Ϫ9, and 10Ϫ11 M) for 24, 36 or 48 h. For time course experiments, the Vector (Promega, Madison, WI). Orientation of the insert was deter- culture medium was changed at the same time, and all cells were mined by DNA sequencing. The cPLA and glyceradehyde 3-phosphate harvested at the same time. Dexamethasone (10Ϫ7 M) was added at the dehydrogenase (GAPDH) RNA probes were prepared by in vitro tran- indicated times prior to harvesting. At the indicated times treated and scription using T7 polymerase with [␣-32P]CTP. The p11 RNA probes control cells were rinsed three times with cold PBS. After washing, the was prepared by in vitro transcription using SP6 polymerase with cells were transferred to 0.5 ml of homogenization buffer; 50 mM Hepes, [␣-32P]CTP. An RPA assay kit (RPAII, Ambion, Austin Texas) was used.
pH 8.0, 1 mM EDTA, 1 mM EGTA, 100 ␮M leupeptin, 1 mM dithiotherei- Hybridization was performed at 45 °C for 16 h and with 10 ␮g (for tol, 10 mM phenylmethylsulfonyl fluoride, 0.5 mM soybean trypsin in- GAPDH) or 20 ␮g (for cPLA ) and 40 ␮g (for p11) of total RNA. 104 dpm hibitor, 15 mM aprotinin, and 0.5% Triton X-100. Cells in homogeniza- (for GAPDH) and 2 ϫ 104 dpm (for cPLA and p11) of 32P-labeled RNA tion buffer were sonicated for 15 s times three using a microprobe. Total probe were used. After hybridization, the unhybridized RNA was di- protein was assayed by BCA reagent (Pierce). Samples containing 20 ␮g gested by addition of 1:100 diluted RNaseA/T1 mix at 37 °C for 60 min.
of cell lysate protein were separated on 18% Tris-glycine gels (Novex, Digestion was terminated by the addition of RNase inactviation and San Diego, CA) using Tris-glycine SDS running buffer. The separated precipitation mixture. The protected RNA fragment was analyzed by proteins were electrophoretically transferred onto a nitrocellulose mem- autoradiography after separation on 6% polyacrylamide, 8 M urea gels brane (Novex), then blocked with 5% non-fat dry milk overnight. p11 protein expression was detected by using 1:2000 dilution of mouse-anti- Effect of RU486 on Dexamethasone-induced p11 Expression—The p11 monoclonal antibody (Transduction Laboratories, Lexington, KY) HeLa cells grown on 175-cm2 flasks were treated with dexamethasone and 1:5000 dilution horseradish-peroxidase-conjugated donkey-anti- (10Ϫ7 M) with or without the glucocorticoid receptor antagonist, RU486 mouse IgG as second antibody (Jackson ImmunoResearch Laboratories, (10Ϫ7, 10Ϫ8, 10Ϫ9, 10Ϫ10, 10Ϫ11, and 10Ϫ12 M) for 24 h. At the end of Inc., West Grove, PA). The blot was developed using the ECL Western incubation time, crude cytosolic extracts of treated and control cells blotting detection system (Amersham Pharmacia Biotech).
were prepared and Western blots were done as described in the exper- Immunoblot of cPLA Protein—HeLa cells grown in 175-cm2 flasks imental procedures section for immunoblot of p11 protein.
were treated with dexamethasone (10Ϫ7, 10Ϫ9, and 10Ϫ11 M) for 24, 36, Arachidonic Acid Release from Dexamethasone-treated Cells—The or 48 h. At the indicated times, crude cytosolic extracts of treated and HeLa cells grown on T-75-cm2 cultured flasks were labeled for 18 h with control cells were prepared as described above for the immunoblot of 1 ␮Ci/ml [5,6,8,9,11,12,14,15-3H]arachidonic acid ([3H]AA)(214 Ci/ p11 protein. Samples containing 20 ␮g of cell lysate protein were mmol; Amersham Pharmacia Biotech) in DMEM media with 10% fetal separated on 8% Tris-glycine gels (Novex) using Tris-glysine SDS run- calf serum. Subsequently, some cultures were treated with dexametha- ning buffer. cPLA protein expression was detected by using 1:1000 M) for 24 h, while others were maintained as controls.
dilution rabbit-anti-human cPLA polyclonal antibody (provided by the Following 20-h incubation with dexamethasone, all cells were relabeled Genetics Institute, Boston, MA) and 1:5000 dilution of horseradish- with 1 ␮Ci/ml [3H]AA for 4 h before harvesting the cells. For studies of peroxidase-conjugated goat-anti-rabbit IgG as a second antibody (Jack- AA release after calcium ionophore stimulation, following three washes, son ImmunoResearch Laboratories, Inc.). The blot was developed using 12 ml of calcium ionophore A23187 (10Ϫ6 M) (Calbiochem) in HBSS(ϩ) the ECL Western blotting detection system.
with 0.5% BSA or HBSS(ϩ) with 0.5% BSA alone were added to each Immunoprecipitation of Native p11 Protein from HeLa and BEAS-2B flask, and the cells were incubated at 37 °C for 30 min. The supernatant Cells—The HeLa or BEAS-2B cells grown on 175-cm2 culture flasks was harvested for HPLC analysis. The samples for HPLC analysis were were washed with cold PBS, and the cells were lysed in 0.5 ml of homogenization buffer with protease inhibitors, but without EGTA and ciates, Milford, MA) and chromatographed by reverse phase HPLC.
EDTA. The crude cytosolic protein was isolated as described above for cartridges were prepared with 15 ml of meth- immunoblot for p11 protein. For immunoprecipitation, the isolated anol followed by 5 ml of 5 mM EDTA and 10 ml of water. Samples were crude cytosolic fraction (200 ␮l, 400 ␮g of protein) was added to a loaded onto the cartridges washed with 10 ml of water and eluted with microcentrifuge tube containing 1 ml HBSS (with calcium and magne- 4 ml of methanol. The methanol fraction was collected and evaporated sium) and 10 ␮l of rabbit anti-human cPLA antibody. The samples to dryness under steady flow nitrogen gas and resuspended in 200 ␮l of were incubated at 4 °C for 30 min, 25 ␮l of Protein G Plus/Protein methanol for analysis by HPLC. An ultrasphere C A-agarose (Pierce) was then added to each sample, and the mixture was mm) (Beckman Instruments) with 5-␮m particle size was used. A gra- incubated at 4 °C for 4 h, followed by centrifugation in a microcentri- dient program was used with mobile phase A, water/acetonitrile/phos- fuge at 2500 rpm for 5 min at 4 °C. The supernatant was aspirated, and phoric acid (75:25:0.025), and mobile phase B, methanol/acetonitrile/ the pellet was washed four times with 1.0 ml cold PBSTDS (phosphate- trifluoroacetic acid (60:40:0.0016), at a flow rate of 1.5 ml/min. The AA buffered saline, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% fraction of HPLC elution was collected and measured for radioactivity.
sodium dodecyl sulfate) with repeated centrifugation. After four wash- Stable Transfection of a p11 Antisense Plasmid in HeLa Cells—A ings with PBSTDS, 20 ␮l of protein loading buffer was added to the 321-bp cDNA corresponding to bases 115– 436 of the p11 cDNA se- pellet and the sample was boiled for 10 min before electrophoresis on quence was used to construct the antisense p11 expression plasmid, 16% polyacrylamide gels (Novex) using Tris-glycine/SDS buffer. The the insert was cloned into the mammalian expression vector separated proteins were electrophoretically transferred onto a nitrocel- pcDNA3.1(ϩ) (Invitrogen) in the antisense orientation, giving rise to lulose membrane blocked with 5% non-fat milk and then probed with a ASp11-pcDNA3.1(ϩ). The identity and orientation of construct was 1:2000 dilution of mouse anti-human p11 monoclonal antibody. The confirmed by DNA sequencing. HeLa cells grown in 175-cm2 flasks blots were then probed with a 1:5000 dilution of horseradish peroxi- were exposed to 120 ␮l of LipofectAMINE Reagent (Life Technologies, dase-labeled donkey anti-mouse IgG and developed by using the ECL Inc.) with 20 ␮g of ASp11-pcDNA3.1(ϩ) plasmid after repeated wash- ing with serum-free DMEM medium. Control cells were transfected Ribonuclease Protection Assay (RPA) for cPLA and p11 mRNA Lev- with pcDNA3.1(ϩ) expression plasmid alone. Cells were exposed to els—The HeLa cells were treated with dexamethasone (10Ϫ7, 10Ϫ9, and the mixture of LipofectAMINE and plasmid for 4 h. Following re- 10Ϫ11 M) for 24 to 48 h. Total cellular RNA was extracted from 175 cm2 moval of the transfection reagent, fresh DMEM with 10% serum and culture flasks by the single step guanidinium thiocyanate-phenol-chlo- 1000 ␮g/ml Geneticin (G418 sulfate) (Calbiochem) was added to each Dexamethasone Alteration of Cellular p11 flask. Subsequent cultures of selected HeLa cells were routinelygrown in the presence of selective pressure. Transfected HeLa cellswere cloned by limiting dilution and clones used for Western blot andAA release.
For [3H]AA release studies, equal numbers of cells transfected with pcDNA3.1(ϩ) vector alone as control, and the cells transfected with thep11 antisense plasmid ASp11-pcDNA3.1(ϩ) were grown in T-75-cm2culture flasks. Cells were labeled for 18 h with 1 ␮Ci/ml [3H]AA inDMEM medium with 10% fetal calf serum and 1000 ␮g/ml Geneticin.
Following repeated washing with media, 12 ml of fresh medium with10% serum and 1000 ␮g/ml Geneticin were added to each flask. Forstudies of AA release after calcium ionophore stimulation, followingrepeated washing with HBSS(ϩ) with 0.5% BSA for three times, 12 mlof calcium ionophore A23187 (10Ϫ6 M) in HBSS(ϩ) with 0.5% BSA orHBSS with 0.5% BSA without A23187 were added to each flask, and thecells were incubated at 37 °C for 30 min. The supernatants were ex-tracted by Sep-Pak C cartridges and chromatographed by reverse phase HPLC as described above. The AA fraction of HPLC elution wascollected and measured for radioactivity.
Stable Transfection of a p11 Expression Plasmid in HeLa Cells—A cDNA containing the coding region of the p11 gene was cloned into the FIG. 1. The effect of dexamethasone on p11 protein levels. A,
mammalian expression vector pcDNA3.1(ϩ) (Invitrogen) to create the effect of dexamethasone on p11 protein levels in HeLa cells. Cells p11-pcDNA3.1(ϩ). The identity and orientation of construct was con- were grown to near confluence and then treated with dexamethasone firmed by DNA sequencing. The pcNDA3.1(ϩ) vector carries the human (10Ϫ7 M) for 24 – 48 h. Cell lysates from treated and untreated cells wereprocessed as described under “Experimental Procedures,” and 20 ␮g of cytomegalovirus immediate early enhancer-promoter sequences to pro- total protein was subjected to gel electrophoresis and immunoblotting.
mote constitutive expression of the cloned p11 insert in mammalian B, the dose effect of dexamethasone on p11 protein levels in HeLa cells.
cells. The HeLa cells grown in 175-cm2 flasks were exposed to 120 ␮l of Cells were grown to near confluence and then treated with dexametha- LipofectAMINE Reagent (Life Technologies, Inc.) with 20 ␮g of sone (10Ϫ7 to 10Ϫ11 M) for 24 h. Cell lysates from treated and untreated p11-pcDNA3.1(ϩ) plasmid after repeated washing with serum-free cells were processed as described under “Experimental Procedures” and DMEM medium. Control cells were transfected with pcDNA3.1(ϩ) ex- 20 ␮g of total protein was subjected to gel electrophoresis and immu- pression plasmid alone. Cells were exposed to the mixture of Lipo- noblotting. C, the effect of dexamethasone on p11 protein levels in fectAMINE and plasmid for 4 h. Following removal of the transfection BEAS-2B cells. Cells were grown to near confluence and then treated reagent, fresh DMEM with 10% serum and 1000 ␮g/ml Geneticin (G418 with dexamethasone (10Ϫ7 M) for 24 – 48 h. Cell lysates from treated and sulfate) (Calbiochem) was added to HeLa cells. Subsequent cultures of untreated cells were processed as described under “Experimental Pro- selected HeLa cells were routinely grown in the presence of selective cedures,” and 20 ␮g of total protein was subjected to gel electrophoresis pressure. Transfected HeLa cells were cloned by limiting dilution and clones were used for Western blot and AA release after four passages.
For [3H]AA release, equal numbers of cells transfected with pcDNA3.1(ϩ) vector alone as control and the cells transfected with the p11 expression plasmid p11-pcDNA3.1(ϩ) were grown in T-75-cm2 cul-ture flasks. Cells were labeled for 18 h with 1 ␮Ci/ml [3H]AA in DMEMmedium with 10% fetal calf serum with 1000 ␮g/ml Geneticin. Forstudies of AA release after calcium ionophore stimulation, followingthree washes with HBSS(ϩ) with 0.5% BSA, 12 ml of calcium ionophoreA23187 (10Ϫ6 M) in HBSS(ϩ) with 0.5% BSA or HBSS(ϩ) with 0.5%BSA alone were added to each flask, and the cells were incubated at37 °C for 30 min. The supernatants were collected, extracted on Sep-Pak C cartridges and chromatographed on reverse phase HPLC as described above. The AA fraction of HPLC elution was collected andmeasured for radioactivity.
Dexamethasone Increases p11 Protein Levels in Human HeLa Cells and BEAS-2B Cells—The effect of dexamethasone treat-ment on human epithelial cell expression of p11 was studied byWestern blot of two different epithelial cell lines, HeLa cells FIG. 2. The effect of dexamethasone on p11 mRNA levels in
and BEAS-2B cells. Fig. 1A demonstrates the effect of dexa- HeLa cells. A, the effect of dexamethasone on p11 mRNA levels. HeLa
methasone treatment of HeLa cells on cellular p11 accumula- cells were treated with dexamethasone (10Ϫ7 M) for 24, 36, and 48 hbefore total RNA was extracted. Ten ␮g and 40 ␮g of the total RNA were tion. Treatment of cells with dexamethasone (10Ϫ7 M) for hybridized to GAPDH and p11-specific radiolabeled cRNA probes, re- 24 – 48 h resulted in a significant increase in p11 protein ex- spectively, and assayed by RPA. The protected fragments of p11 (320 pression in cell lysates. In addition, treatment of cells with bp) and GAPDH were visualized by autoradiography. The result shown is representative of three separate experiments demonstrating the same result. B, the dose effect of dexamethasone on p11 mRNA levels.
dose-related increase cellular p11 protein levels (Fig. 1B).
The HeLa cells were treated with dexamethasone (10Ϫ7, 10Ϫ9, and Treatment of BEAS-2B cells with dexamethasone (10Ϫ7 M) for 10Ϫ11 M) for 24 h before total RNA was extracted. Ten ␮g or 40 ␮g of the 24 – 48 h also resulted in a significant increase in p11 protein total RNA were hybridized to GAPDH and p11-specific radiolabeled expression in cell lysates (Fig. 1C).
RNA probes and assayed by RPA. The protected fragments of p11 (320bp) were visualized by autoradiography. The result shown is represent- Effect of Dexamethasone on Steady State Levels of p11 ative of three separate experiments.
mRNA—Steady state levels of mRNA for p11 were measuredby RPA of total cellular RNA extracted from HeLa cells thatwere incubated without or with dexamethasone (10Ϫ7 M) for related change in p11 mRNA levels (Fig. 2B).
24 – 48 h. As shown in Fig. 2A, these cells produce p11 mRNA Effect of Dexamethasone on cPLA Protein and mRNA Levels and the steady state level of p11 mRNA was increased by in HeLa Cells—The effect of dexamethasone treatment on hu- dexamethasone treatment over 24 – 48 h. In addition, dexa- man epithelial cell expression of cPLA was studied by Western methasone in concentrations of 10Ϫ7 to 10Ϫ11 M induced a dose- blot of cell lysates. Treatment of cells with dexamethasone Dexamethasone Alteration of Cellular p11 FIG. 4. Immunoprecipitation of p11⅐cPLA complex. A, immuno-
precipitation of the p11⅐cPLA complex from HeLa cells. Cell lysates from untreated cells and cells treated with dexamethasone (10Ϫ7 M) for24, 36, and 48 h were incubated with rabbit anti-human cPLA , 25 ␮l of Protein G Plus/Protein A-agarose beads was then added to each samplefor further incubation. The beads were collected, washed, and subjectedto SDS-polyacrylamide gel electrophoresis. The precipitated p11 pro-tein was then detected by Western blotting analysis as described under“Experimental Procedures.” The position of p11 protein is indicated. B,immunoprecipitation of the p11⅐cPLA complex from BEAS-2B cells.
Lysates from untreated cells and cells treated with dexamethasone (10Ϫ7 M) for 24, 36, and 48 h were incubated with rabbit anti-human cPLA , and 25 ␮l of Protein G Plus/Protein A-agarose beads was then IG. 3. The effect of dexamethasone on cPLA
protein and
steady state mRNA levels. A, the effect of dexamethasone on cPLA
added to each sample for further incubation. The beads were collected, protein levels in HeLa cells. Cells were grown to near confluence and washed, and subjected to SDS-polyacrylamide gel electrophoresis. The precipitated p11 protein was then detected by Western blotting analysis from treated and untreated cells were processed as described under as described under “Experimental Procedures.” The position of p11 “Experimental Procedures,” and 20 ␮g of total protein was subjected to gel electrophoresis and immunoblotting. B, the dose effect of dexa-methasone on cPLA protein levels in HeLa cells. Cells were grown to In an attempt to determine if the effect of dexamethasone on near confluence and then treated with dexamethasone (10Ϫ7 to 10Ϫ11 M)for 24 h. Cell lysates from treated and untreated cells were processed as p11 protein levels is mediated via a glucocorticoid receptor described under “Experimental Procedures,” and 20 ␮g of total protein interaction, RU486 (10Ϫ7 to 10Ϫ12 M) was incubated with cells was subjected to gel electrophoresis and immunoblotting. C, the effect of prior to and concomitant with the dexamethasone treatment.
dexamethasone on cPLA mRNA levels. HeLa cells were treated with Treatment with RU 486 resulted in a dose-dependent inhibi- dexamethasone (10Ϫ7 M) for 24, 36, and 48 h before total RNA wasextracted. Ten ␮g and 20 ␮g of the total RNA were hybridized to tion of the dexamethasone-induced increases in p11 protein GAPDH and cPLA -specific radiolabeled cRNA probes, respectively, levels. Fig. 5 shows the effect of RU486 (10Ϫ10-10Ϫ12 M) on and assayed by RPA. The protected fragments of cPLA (306 bp) and dexamethasone-induced p11 protein levels.
GAPDH were visualized by autoradiography. The result shown is rep- Dexamethasone Inhibits AA Release from the HeLa Cells— resentative of three separate experiments.
The results from RPA and Western blot studies indicated thatdexamethasone treatment had an effect on p11 mRNA levels (10Ϫ7 M) for 24 – 48 h had no effect on cPLA protein expression and protein production but little or no effect on the mRNA (Fig. 3A). In addition, treatment of cells with 10Ϫ7, 10Ϫ9, and expression or protein level of cPLA . In these cells, dexametha- 10Ϫ11 M dexamethasone for 24 h did not result in a change in sone treatment does alter the release of 3H-labeled AA both at cPLA protein levels (Fig. 3B). The effect of dexamethasone base line and after exposure to the calcium ionophore A23187.
treatment on human epithelial cell expression of cPLA was Fig. 6 demonstrates that labeled AA release from dexametha- also studied by RPA of HeLa cells treated with dexamethasone.
sone-treated HeLa cells (HD) is lower than that from untreated Fig. 3C demonstrates the effect of dexamethasone treatment of HeLa cells (HC). After treatment with A23187, the release of HeLa cells on steady state levels of cPLA mRNA. Treatment of labeled AA from dexamethasone-treated HeLa cells (HDϩA) is cells with dexamethasone (10Ϫ7 M) for 24 – 48 h had no clear significantly decreased compared with untreated control cells Dexamethasone Increases p11 Bound to cPLA —The above Antisense Inhibition of p11 Increases AA Release—We have results demonstrated that dexamethasone treatment in- shown that dexamethasone increases p11 expression and in- creased p11 expression, but had little or no effect on cPLA hibits PLA activity in vitro. It has been reported that p11 can expression. To further investigate the interaction between bind to cPLA and inhibit cPLA activity in vitro. In order to p11 and cPLA in human epithelial cells, immunoprecipita- study whether dexamethasone might alter cPLA activity in tion of the p11⅐cPLA complex from HeLa cells and BEAS-2B part by increasing p11 expression in human cells, we per- cells was performed. As shown in Fig. 4, A and B, p11 was formed two studies. First, we constructed a p11 antisense plas- precipitated from the HeLa cell and BEAS-2B cell lysates by mid and then stably transfected HeLa cells to examine the AA rabbit anti-human cPLA polyclonal antibody followed by the release in these cells. Western blot studies of cloned trans- addition of Protein G Plus/Protein A-agarose. Immunoblots of formed cells showed that p11 protein production was decreased the purified complex were developed for p11 protein. There in HeLa cells which were transfected with ASp11-pcDNA3.1(ϩ) was more p11 coprecipitated with cPLA after dexametha- plasmid compared with HeLa cells, which were transfected sone treatment. This result demonstrated that dexametha- with pcDNA3.1(ϩ) plasmid alone (Fig. 7A). There was no sone treatment resulted not only in an increase in cellular change in cPLA expression in these cells (Fig. 7B). [3H]AA p11 protein but also in an increase in p11 bound to cPLA .
release from the HeLa cells that were permanently transfected RU486 Inhibits Dexamethasone-induced p11 Protein Increases— with p11 antisense plasmid was increased both at base line and Dexamethasone Alteration of Cellular p11 FIG. 5. The effect of RU486 treatment on dexamethasone-in-
duced p11 protein levels. Cells were grown to near confluence.
RU486 was incubated with cells prior to and concomitant with or
without dexamethasone (10Ϫ10 to 10Ϫ12 M) for 24 h. Cell lysates from
treated and untreated cells were processed as described under “Exper-
imental Procedures,” and 20 ␮g of total protein was subjected to gel
electrophoresis and immunoblotting.
FIG. 7. The p11 and cPLA protein levels in HeLa cells trans-
fected with a p11 antisense plasmid. A, cell lysates from cells
transfected with the p11 antisense plasmid or cells transfected with a
control vector were processed as described under “Experimental Proce-
dures,” and 20 ␮g of cell lysate protein was subjected to gel electro-phoresis and immunoblotting for p11 protein. Three different samplesof cell lysates of p11 antisense cells and control cells were processed. B,protein levels of cPLA in HeLa cells transfected with a p11 antisense plasmid. Cell lysates from cells transfected with the p11 antisenseplasmid or cells transfected with a control vector were processed as FIG. 6. [3H]AA release from dexamethasone treated HeLa cells.
described under “Experimental Procedures,” and 20 ␮g of cell lysate The cells grown in T-75-cm2 flasks were labeled for 18 h with 1 ␮Ci/ml protein was subjected to gel electrophoresis and immunoblotting for [3H]AA in 12 ml of DMEM medium and then treated with 10Ϫ7 M dexamethasone for 24 h. After repeated washing, the cells were thenincubated with 10Ϫ6 M ionophore A23187 in 12 ml of HBSS (containing1.3 mM Ca2ϩ) for 30 min, and the supernatants were extracted bySep-Pak C cartridges and chromatographed by HPLC as described under “Experimental Procedures.” Data were expressed as AA releasemeasured separately from 11–12 individual flasks from two separatesets of experiments. HC ϭ HeLa control cells; HD ϭ HeLa cells treatedwith dexamethasone; A ϭ treatment with A23187. p Ͻ 0.001 for HCversus HD; p Ͻ 0.001 for HCϩA versus HDϩA.
after exposure to the calcium ionophore A23187 compared withcontrol cells (Fig. 8).
Increased p11 Expression Inhibits AA Release—In order to determine whether dexamethasone inhibition of cellular PLA2activity might be related in part to increasing p11 expression inhuman epithelial cells, we constructed a p11 expression plas-mid and stably transfected HeLa cells to examine the effect ofincreased p11 expression on AA release in these cells. The FIG. 8. [3H]AA release from HeLa cells transfected with a p11
antisense plasmid. The cells grown in T-75-cm2 flasks were labeled
effect of the p11 expression plasmid on cellular p11 protein is for 18 h with 1 ␮Ci/ml [3H]AA in 12 ml of DMEM with Geneticin. After demonstrated in Fig. 9A. Western blot results showed that p11 repeated washing, some cells were then incubated with 10Ϫ6 M iono- protein production was increased in HeLa cells that were phore A23187 in 12 ml of HBSS (containing 1.3 mM Ca2ϩ) or with HBSS transfected with p11-pcDNA3.1(ϩ) plasmid compared with without A23187 for 30 min, and the supernatants were extracted by HeLa cells that were transfected with pcDNA3.1(ϩ) plasmid cartridges and chromatographed by HPLC as described under “Experimental Procedures.” Data were expressed as AA release alone. There was no change in cPLA expression (Fig. 9B). AA measured separately from 10 to 12 individual flasks in each group.
release from the HeLa cells which were permanently trans- VC ϭ vector control cells; ASp11 ϭ HeLa cells transfected with a fected with p11 expression plasmid was decreased both at base plasmid expressing p11 antisense mRNA; A ϭ HeLa cells treated with line and after exposure to the calcium ionophore A23187 (Fig.
A23187. p Ͻ 0.001 for VC versus ASp11; p Ͻ 0.05 for VCϩA versusASp11ϩA.
10). Therefore, dexamethasone treatment increases p11 pro-tein expression and reduces cellular arachidonate release. Fur-thermore, increasing cellular p11 protein production independ- the two EF hand motifs (14, 15). Instead, p11 is present in a ent of dexamethasone treatment reduces cellular AA release as variety of cells separately or as a heterotetramer binding to annexin II. The heterotetramer is composed of two copies of the36-kDa heavy chain, annexin II subunits and two copies of 11-kDa light chain, p11 subunits as (p36) (p11) (32, 33).
p11, or calpactin light chain, is a member of the S-100 family Glucocorticosteroids are potent anti-inflammatory agents.
small calcium binding proteins; however, it has several unique This anti-inflammatory effect may be produced via a variety of features. S-100 proteins contain two EF hands that function as mechanisms. A group of structurally related, calcium-depend- calcium binding domains (13). p11 does not have the ability to ent phospholipid-binding proteins, annexins, which were for- bind Ca2ϩ ions due to amino acid deletions and substitutions in merly known as lipocortins or calpactins, had been shown to be Dexamethasone Alteration of Cellular p11 between the 14-kDa PLA and annexins. Instead, this inhibi- tion may be dependent on the concentration of substrate (34,35), the extent of inhibition being more closely related to theinhibitor:substrate rather than the inhibitor:enzyme ratio. Inaddition, glucocorticoid treatment suppresses the induction ofGroup II sPLA expression in a variety of cells (36 – 40).
cPLA selectively hydrolyzes AA from the sn-2-ester bond of membrane phospholipids. cPLA may play an important role in the production of free fatty acids and lysophospholipids, pre-cursors of eicosanoids and PAF, all of which may function asintracellular second messengers or potent inflammatory medi-ators (1, 3, 5). It has been reported that dexamethasone treat-ment reduces changes in cPLA protein and mRNA levels in- duced by TNF treatment of HeLa cells (41). Dexamethasonemay have other effects on AA metabolism and at earlier timepoints, including effects perhaps not requiring transcriptionsuch as inhibition of phosphorylation of cPLA (42). We did not document an effect of dexamethasone on unstimulated expres-sion of cPLA ; however, we did note an effect of dexamethasone on cellular p11 protein and mRNA levels. Because it has beendemonstrated that p11 can directly interact with the carboxylregion of cPLA and inhibit its activity in vitro (31), we hypoth- esized that a part of the effect of dexamethasone on cellular AA release might be mediated by a dexamethasone-induced FIG. 9. The p11 and cPLA protein levels in HeLa cells trans-
fected with a p11 expression plasmid. A, cell lysates from cells
Four lines of evidence suggest that dexamethasone may alter transfected with the p11 expression vector or cells transfected with cellular arachidonate release in part by induction of p11 pro- control vector were processed as described under “Experimental Proce- tein expression. First, studies in two different cell lines dem- dures,” and 20 ␮g of total protein was subjected to gel electrophoresisand immunoblotting for p11 protein. Two cell lysates from cells trans- onstrate that dexamethasone induces human epithelial cell fected with the p11 expression vector and cells transfected with the p11 gene expression and protein production. This effect was not control vector were processed. B, protein levels of cPLA in HeLa cells associated with a reduction of cPLA expression in HeLa cells.
transfected with a p11 expression vector or cells transfected with a RU486, an antagonist that competes with glucocorticoids for control vector. Cell lysates from cells transfected with the p11 expres-sion or cells transfected with a control vector were processed as de- binding to the glucocorticoid receptor (43, 44), blocked the scribed under “Experimental Procedures,” and 20 ␮g of cell lysate stimulatory effect of dexamethasone on p11 protein production, protein was subjected to gel electrophoresis and immunoblotting for suggesting that dexamethasone-induced p11 gene expression and subsequent protein synthesis occurs via a glucocorticoidreceptor-mediated pathway. Second, in dexamethasone-treatedcells, there was increased p11 binding to cPLA as evidenced by p11⅐cPLA complex. Third, cells stably transfected with a plas- mid that expresses p11 antisense mRNA and that subse-quently express less p11 protein have enhanced release ofprelabeled AA both at base line and after stimulation with theionophore A23187. Fourth, we studied the effect of p11 on AArelease in the setting of overexpression of p11 protein in ahuman epithelial cell line, Hela cells. The release of prelabeledAA from cells that overexpressed p11 was significantly lowerthan that from control cells. Therefore, overexpression of p11inhibits PLA activity and reduces the release of AA from [3H]AA-prelabeled cells. Thus, manipulation of p11 levels in-dependent of corticosteroid therapy also alters AA release from FIG. 10. [3H]AA release from HeLa cells transfected with a p11
expression plasmid. The cells grown in T-75-cm2 flasks were labeled
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