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EGF-induced PGE2 release is synergistically enhanced in retinoic acid treated fetal rat lung cells
Vol. 162, No. 3, 1989 August
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
15, 1989
1515-1521
EGF-Induced PGE; Release is Synergistically Enhanced in Retinoic Acid Treated Fetal Rat Lung Cells Kerby C. Oberg* and Graham Carpenter Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146
Received
June
12,
1989
SW Retinoic acid has been shown to induce a 25fold increase in?-EGF binding capacity thiough increased EGF receptor synthesis in a fetal rat lung (FRL) cell line (1). In FRL cells, incubation with either EGF or retinoic acid induces a modest increase in PGE, secretion (80% or 40%, respectively). However, in the presence of both EGF and retinoic acid, FRL cells exhibit a 6.4-fold increase in PGE, secretion. Retinoic acid and EGF dose-response curves demonstrate that the effect on PGE, secretion correlates with the retinoic acid induced increase in EGF receptors. These data suggest a relationship between increased EGF receptor expression and increased EGF responsiveness. Furthermore, these data indicate a potential mechanism by which EGF and retinoic acid may interact in lung physiology. 0 1989 Academic Press, Inc.
Epidermal mitogenic
growth factor (EGF), a small polypeptide
effects, initiates
its cellular
(6045 daltons) known for its
responses by binding
to a 170,000 dalton
transmembrane receptor on the surface of target cells. The expression of this receptor in fetal rat lung cells can be enhanced by retinoic acid, a metabolite of vitamin A (1). EGF accelerates cellular
proliferation
of lung tissue (2-4) and is associated with increased
surfactant phospholipids (5). Furthermore, vitamin A has been reported to enhance lung tissue proliferation and differentiation (6), and also to be essential in maintenance of the differentiated
state of lung epithelia
(7). However, the mechanisms by which EGF and
retinoic acid may interact to influence lung development
has yet to be resolved.
Although EGF is best known for its mitogenic effects, the hormone also illicits a host of other responses that appear unrelated to mitogenicity (for a review see 8). One such response that is reported for certain cells is the EGF-induced
*Current address: Department Loma Linda, CA 92350.
production of prostaglandins
of Anatomy, Loma Linda University
School of Medicine,
0006-291X/89
1515
$1.50
Copyright 5 1989 by Acadenaic Press, Inc. All rights of reproduction in any jimn reserved.
Vol. 162, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(9-13). This is nearly always reported as an increase in PG&. have the capacity to stimulate the release of pulmonary
Interestingly,
prostaglandins
surfactant (14 & 15). Thus, we
chose to investigate the effects of retinoic acid and EGF on PGE$ secretion in cultured fetal lung cells. The following experiments were performed to determine if the changes in EGF receptor expression induced by retinoic acid could be correlated with changes in EGFinduced prostanoid synthesis.
MATERIALS
AND METHODS
A. Tissue Culture Cultures of fetal rat lung cells (16), an epithelial cell designated FRL, were grown in a humidified incubator at 37 C with 5% in Dulbecco’s modified Eagles medium (DMEM)(GIBCO) supplemented with 10% fetal bovine serum (FBS “4 (GIBCO) and 20 mM HEPES (Calbiochem). Cells used for experiments were plated into multi-well dishes (Costar), at a density of 6,000 cells per cn?, 510% confluence, in DMEM plus 10% calf serum (CS)(GIBCO) unless otherwise noted. The cells were used for experiments when the cells were approximately 8090% confluent. Retinoic acid (Sigma)(or vehicle - 50% EtOH) was added, as described from a stock solution (retinoic acid - 500 PM in 50% EtOH). Cell counts were made by tryp&kiq duplicate wells, and electronically counting (Coulter Instruments, Inc.). B. Receptor Bindiug Assays Mouse-derived EGF was isolated (17) and radiolabeled with ‘=I - as previously described (18). A complete description of the binding assaymethods employed has been published elsewhere (19). In brief, cells were washed twice and DMEM plus 0.2% bovine serum albumin (BSA)(GIBCO) was added. To measure surface binding capacity, the cells were chilled to @C on au ice bath for 20 mm and then incubated with 25 rig/ml (unless otherwise noted) ‘=I-EGF for 3 hrs at 8C. Non-specilic binding was determined by adding a 40-fold excess of unlabeled EGF to parallel sample wells. Excess labeled hormone was removed with 3 washes of ice-cold Hank’s balanced salt solution (HBSS) plus 0.2% BSA. Total cell-associated radioactivity was determined by incubation at 37”. The cells were then solubiid in 1N NaOH and the radioactivity in the samples was measured with a gamma counter (Beckman). Counts were normalized to cpm per 18 cells. Data points represent the mean of triplicate samples with nonspecific binding subtracted. C. PGE Assay %‘I edia were collected from cells incubated in serum-free medium in the presence or absence of retinoic acid and EGF for various lengths of time. The samples were assayed as descriid in NEN’s PG RIA kit. Briefly, 100 ~1 duplicates of samples and standards were transferred to 4 ml polyethylene tubes. ?- 3 G (100 ~1) was added to each tube and vortexed. PGl$ antibody (100 ~1) was added to each tube (except the bl 2k and tubes to identify the specific activity of the radiolabeled PGEJ and vortexed. The samples were incubated overnight in a refrigerator. One ml of precipitating reagent (polyethylene glycol mw 6,000) was added to each tube (except tubes for specific activity) and vortexed. The samples were incubated for another 20-u) min on ice, then centrifuged at 2500 x g for 30 min in a chilled swinging bucket centrifuge. The supernate was discarded and the radioactivity of the pellet was measured by a gamma counter. The PG in each tube was calculated from the standard curve generated. Each data point represents the average of 2 uplicate assays.
RESULTS
AND DISCUSSION
For some time it has been known that retinoic acid can alter the ‘=I-EGF binding capacity in certain cells (20), although, it has been difficult to correlate the alterations in binding capacity with any physiologic actions of EGF. Komura ti & (21) associated retinoic acid-induced increase in EGF-stimulated thymidine incorporation with increased ‘*‘I-EGF binding, yet other investigators have not been able to duplicate this observation in other cell types (22). The effect of retinoids on other EGF-induced responses, such as prostanoid secretion, has yet to be determined. A fetal rat lung cell line (FRL) was chosen to study the effects of retinoic acid on EGF responses, because both agents appear to 1516
BIOCHEMICAL
Vol. 162, No. 3, 1989
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PH EGF
+
Agent Untreated Retinoic Acid
5 7
+ 7.62 7.40
9 45
7.28 6.78
Cells were incubated for 24 hrs in the presence or absence of retinoic acid (5 PM). The cells were rinsed and incubated for another 24 hrs in serum-free media in the absence or presence of EGF (25 rig/ml) and retinoic acid. The media was then collected, the pH measured and duplicate 100 ~1 aliquot assayed for PGE, content. The PGE, values represent the average of the duplicates.
influence lung maturation (2-7). Furthermore, retinoic acid has been shown, in these cells, to stimulate a 3-fold increase in the expression of EGF receptors (1). To determine
the effects of retinoic
acid and EGF on prostaglandin
secretion,
confluent FRL cells were preincubated for 24 hrs in the presence or absence of retinoic acid (5 PM). One-half of each group was then stimulated with EGF (25 rig/ml) for 24 hrs and the medium from each sample was assayed, by a radioimmuno
assay (RIA), for PGS.
The results (Table 1) indicate that without retinoic acid preincubation, FRL cells secreted only a slightly higher level (approximately 80%) of PGE, in response to EGF addition. However, EGF addition interacts synergistically with retinoic acid pretreatment
of FRL cells
inducing a 6.4-fold increase in PGE, secretion. Interestingly,
a decrease in media pH that paralleled the level of PGE, secretion was
observed in cultures incubated in the absence or presence of retinoic acid and/or EGF (Table 1). Since prostaglandins are often released in response to injury, it could be argued that the acidic conditions
signaled the injury response producing prostaglandins.
This
notion, however, does not seem likely because acidic media (pH 6.9), altered by .l % HCl (lM), did not increase PGE, secretion when incubated with untreated FRL cells (data not shown).
These results suggest that although altered pH may be associated with PGS
release, it is not the cause. In retinoic acid treated FRL cells, increased levels of PGE, can be detected in the medium 2 hrs after EGF addition, nonetheless, the differential influence of EGF on control and retinoic acid treated cells becomes more pronounced as PGE, levels accumulate over a 48 hr time course (Figure 1). To determine whether the increase in PGE, secretion was associated with increases in EGF binding capacity, parallel cultures of cells treated with increasing concentrations of 1517
Vol. 162, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
120
I
.a* ’ unboatoo -O- +EGF -*a-. + R&ii + Reb +
, A& Acid & EGF
/’
/
,’
/
/
/’
1”
/ /’
<
0
........_..*............-......... .eo *..................._....................
I
I
I
12
24
36
..
. ... ...... ......’
‘I”nneAfter EGF AdUtii (hs)
Frc;uaEl. ~~~C-OFEGF--~-OPPG~~-ACID ‘lkxnur Fl2.L m Cells were pretreated for 24 hrs in the presence or absence of retinoic acid (5 PM). The cells were washed twice and refed with serum-free media (retinoic acid wasalso re-added to appropriate cultures) and incubated in the presence or absence of EGF (25 rig/ml). At the indicated times the media from specified samples wascollected and frozen. After all of the samples were collected the media was thawed and duplicate 100 ~1aliquot were assayedfor the presence of PGE;.
retinoic acid for 24 hrs were analyzed for either PGE, secretion or ‘=I-EGF binding capacity. The results demonstrate a retinoic acid concentration-dependent correlation between retinoid induced increases in”= I-EGF binding capacity and EGF-stimulated PGE, secretion (Figure 2). Retinoic acid concentrations of l-10 PM produced maximal increases in both 125I-EGF binding and PGE, secretion. Similarly, in cells treated with retinoic acid and then incubated with increasing concentrations of EGF or ‘%EGF, respectively, there were similar increases in the level of PGE, secreted or radiolabeled ligand binding (Figure 3). Maximal potentiation of PGE, secretion by EGF required concentrations of the growth factor sufficient to achieve near saturation of binding. These data demonstrate a correlation between the extent of EGF receptor occupancy and this EGF-induced response. Collectively, the results of both dose-response experiments suggest a relationship between the retinoic acid-induced increase in EGF receptor numbers and EGF responsiveness, although the influence of retinoic acid on other aspects of cell metabolism 1518
Vol. 162, No. 3, 1989
BIOCHEMICAL
Retinoic
0.0
aAcId
L\\
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
bnc+dMr)
I
c
I
10 -09
10
-
10
I
1
10 a
-07
10
-05
10 -a
Retinoic Acid (moles/liter)
FIGURE 2.
ACID CON -TION ON EGF-STIMULMED ml!+s REIUSEL Cellswere incubatedwith various concentrationsof retinoic acid for 24 hrs. The cell were then washedand refed with serum-freemedia,EGF (25 rig/ml) and the appropriate concentrationof retinoic acid. After incubation for another 24 hrs, the media was collected and assayedfor the presenceof PGQ. (Inset) Effect of various retinoic acid concentrationson ‘=I-EGF binding. 125
Gi 4 rn = 8 0
TEE Eppecr OF R~INOIC
I
28,
retinoic
100
0 75 F5 0. -0 g50 iirn
9
0
20
40
60 so EGF hglml)
acid treated
loo
120
Retinoic Acid
f N 25
0
10
20
30
40
50
EGF (rig/ml)
FIGDRE 3.
PGE, !hCREIlON: EGF DOSE-REsmNsrr.~~J-IWEM &IINOIC Acm m ZEUS. Cellswere incubatedwith retinoic acid (5 PM) for 24 hrs. The cell were then washedand refed with serum-freemedia, EGF at various concentrationsand retinoic acid (in the appropriatecultures). After incubationfor another 24 hrs, the media was collected and assayedfor the presenceof PGQ. (Inset) ‘?-EGF binding as a function of EGF concentration.
%EA’lICD
1519
Vol.
162,
No.
3, 1989
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
that could potentiate the effects of EGF can not be ruled out. Irrespective of the mechanism, the observation that retinoic acid and EGF interact synergistically to enhance PGE, release is significant.
Since PGE, has been shown to enhance pulmonary surfactant
secretion (14 & 15) these data may indicate a mechanism by which retinoic acid and EGF participate
in lung function.
ACKNOWLEDGMENTS We thank Kelly Adair for excellent technical assistance. This research was supported by grants HI-14214 from the NIH and a grant from the Walter E. Macpherson Society.
REFERENCES 1. Oberg KC, Soderquist AM, Carpenter G. (1988). Accumulation of EGF receptors in retinoic acid treated fetal rat lung cells is due to enhanced receptor synthesis. Molec Endocrinol2:959-%5. 2. Goldin GV, Opperman LA. (1980). Induction of supernumerary tracheal buds and the stimulation of DNA synthesis in the embryonic chick lung and trachea by epidermal growth factor. J Embry ExptI Morph
60235243.
3. Sundeii HW, Gray ME, Serenius FS, Escobedo MB, Stahhnan MT (1979). Effects of epidermai growth factor on lung maturation in fetal lambs. Am J Path01 100:707-726. 4. Catteron WZ, Escobedo MB, Sexson WR, Gray ME, SundeU HW, Stahhnan MT. (1979). Effect of epidermal growth factor on lung maturation in fetal rabbits. Pediat Res 133104-108. 5. Haigh R, D’Souza SW, MickIewright L, Gregory H, Butler SJ, HoUingsworth M, Domtai P, Boyd RDH. (1989). Human amniotic fluid urogastrone (epidermal growth factor) and fetal lung phosphohpids. British J Ob Gyn %:171-178. 6. Klann RC, Marchok AC. (1982). Effects of retinoic acid on ceii proliferation and ceil differentiation in a rat tracheal epithelial cell line. Ceil Tissue Kinet 153473482. 7. Chytii F. (1985). Vitamin A and lung development. Pediatr Puhnonol l(supp1): sll5-~117. 8. Carpenter G, Wahl MI. (1989). The epidermal growth factor family of mitogens. In: Handbook of experimental pharmacology: Peptide growth factors and their receptors. Spom M, Roberts A. (ed) (ii press). 9. Takasu N, Sato S, Yamada T, Shimii Y. (1987). EpidermaI growth factor (EGF) and tumor promoter 12-0-tetradecanoylphorbol U-acetate (TPA) stinndate PG synthesis and thymidine incorporation in cultured porcine thyroid cells. Biochem Biophys Res Comm 143:880-884. 10. Lorenzo JA, Quinton J, Sousa S, Raisz LG. (1986). Effects of DNA and prostagIandin synthesis inhibitors on the stimulation of bone resorption by epidermal growth factor in fetal rat long-bone cultures. J CIin Invest 77:1&97-19(X. 11. Muramatsu I, Hollenberg MD, Lederis K. (1985). Vascular actions of epidermai growth factor-urogastrone: possible relationship to prostaglandm production. Can J Physiol Pharmacol63:994-999. 12. Feyen JH, van der Wilt G, Moonen P, Di Bon A, Nijweide PJ. (1984). Stimulation of arachidonic acid metabolism in primary cuitures of osteoblast-Iike cells by hormones and drugs. ProstagIandii 28:769-781. 13. Warner MR, Rappaport prostagIandin--mediated
MS, Krieger NS, Novak RF, Stem PH. (1984). Ametantrone resorption in bone organ culture. Prostagiandins 28:469-476.
inhibits
14. Kitterman JA, Liggins GC, Clements JA, Campos GA, Lee CH, Bahard PL. (1981b). Inhibitors prostagiandin synthesis, tracheal fluid and surfactant in fetal lambs. J Appl Physiol51:1562-1567. 1520
of
Vol. 162, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
15. Marino PA, Rooney SA. (1980). Surfactant secretion in a newborn rabbit lung slice model. Biochim Biophys Acta 620509-519.
17. SaMge CR, C&en S.(1972).Epidcrmalgrowthti
anda newdmivatbe.Rapidkolationprocedure aad chemicalcharacZerlzath J Bid Chem247z7609-7611.
‘%abeled hmuanepidermal growthiaetor(hEGF):Binding,internakation 18. CarpenterG, CohenS.(1976). anddegradation in human&roblasts. J CellBiol7kJ.59-171. 19. JettenAM. (1980).R&bids s&fkUy 2843626
enhance thenumberof epidermal growthfaacartceptora.Nature
20. KomuraE-i,WakimotoH, CJJU-Fung C, TerakawaN, Aono T, Tankawa0, MatusmotoK. (1986).Rctinoic acidenhances cellresponses to epidenual growth factor in moasc mamma3y glad in culture. Endooiaol lm-1536. 21. Kukita T, Youshikawa I-X,OhsakiY, NagataK, Ku&u JC.(1987).E&&s of retinoica&i on tteptors for epidermal growth factor in moue palatal mestchymal cells in vitro. Arch Oral Bil32563.
1521
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
15, 1989
1515-1521
EGF-Induced PGE; Release is Synergistically Enhanced in Retinoic Acid Treated Fetal Rat Lung Cells Kerby C. Oberg* and Graham Carpenter Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146
Received
June
12,
1989
SW Retinoic acid has been shown to induce a 25fold increase in?-EGF binding capacity thiough increased EGF receptor synthesis in a fetal rat lung (FRL) cell line (1). In FRL cells, incubation with either EGF or retinoic acid induces a modest increase in PGE, secretion (80% or 40%, respectively). However, in the presence of both EGF and retinoic acid, FRL cells exhibit a 6.4-fold increase in PGE, secretion. Retinoic acid and EGF dose-response curves demonstrate that the effect on PGE, secretion correlates with the retinoic acid induced increase in EGF receptors. These data suggest a relationship between increased EGF receptor expression and increased EGF responsiveness. Furthermore, these data indicate a potential mechanism by which EGF and retinoic acid may interact in lung physiology. 0 1989 Academic Press, Inc.
Epidermal mitogenic
growth factor (EGF), a small polypeptide
effects, initiates
its cellular
(6045 daltons) known for its
responses by binding
to a 170,000 dalton
transmembrane receptor on the surface of target cells. The expression of this receptor in fetal rat lung cells can be enhanced by retinoic acid, a metabolite of vitamin A (1). EGF accelerates cellular
proliferation
of lung tissue (2-4) and is associated with increased
surfactant phospholipids (5). Furthermore, vitamin A has been reported to enhance lung tissue proliferation and differentiation (6), and also to be essential in maintenance of the differentiated
state of lung epithelia
(7). However, the mechanisms by which EGF and
retinoic acid may interact to influence lung development
has yet to be resolved.
Although EGF is best known for its mitogenic effects, the hormone also illicits a host of other responses that appear unrelated to mitogenicity (for a review see 8). One such response that is reported for certain cells is the EGF-induced
*Current address: Department Loma Linda, CA 92350.
production of prostaglandins
of Anatomy, Loma Linda University
School of Medicine,
0006-291X/89
1515
$1.50
Copyright 5 1989 by Acadenaic Press, Inc. All rights of reproduction in any jimn reserved.
Vol. 162, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(9-13). This is nearly always reported as an increase in PG&. have the capacity to stimulate the release of pulmonary
Interestingly,
prostaglandins
surfactant (14 & 15). Thus, we
chose to investigate the effects of retinoic acid and EGF on PGE$ secretion in cultured fetal lung cells. The following experiments were performed to determine if the changes in EGF receptor expression induced by retinoic acid could be correlated with changes in EGFinduced prostanoid synthesis.
MATERIALS
AND METHODS
A. Tissue Culture Cultures of fetal rat lung cells (16), an epithelial cell designated FRL, were grown in a humidified incubator at 37 C with 5% in Dulbecco’s modified Eagles medium (DMEM)(GIBCO) supplemented with 10% fetal bovine serum (FBS “4 (GIBCO) and 20 mM HEPES (Calbiochem). Cells used for experiments were plated into multi-well dishes (Costar), at a density of 6,000 cells per cn?, 510% confluence, in DMEM plus 10% calf serum (CS)(GIBCO) unless otherwise noted. The cells were used for experiments when the cells were approximately 8090% confluent. Retinoic acid (Sigma)(or vehicle - 50% EtOH) was added, as described from a stock solution (retinoic acid - 500 PM in 50% EtOH). Cell counts were made by tryp&kiq duplicate wells, and electronically counting (Coulter Instruments, Inc.). B. Receptor Bindiug Assays Mouse-derived EGF was isolated (17) and radiolabeled with ‘=I - as previously described (18). A complete description of the binding assaymethods employed has been published elsewhere (19). In brief, cells were washed twice and DMEM plus 0.2% bovine serum albumin (BSA)(GIBCO) was added. To measure surface binding capacity, the cells were chilled to @C on au ice bath for 20 mm and then incubated with 25 rig/ml (unless otherwise noted) ‘=I-EGF for 3 hrs at 8C. Non-specilic binding was determined by adding a 40-fold excess of unlabeled EGF to parallel sample wells. Excess labeled hormone was removed with 3 washes of ice-cold Hank’s balanced salt solution (HBSS) plus 0.2% BSA. Total cell-associated radioactivity was determined by incubation at 37”. The cells were then solubiid in 1N NaOH and the radioactivity in the samples was measured with a gamma counter (Beckman). Counts were normalized to cpm per 18 cells. Data points represent the mean of triplicate samples with nonspecific binding subtracted. C. PGE Assay %‘I edia were collected from cells incubated in serum-free medium in the presence or absence of retinoic acid and EGF for various lengths of time. The samples were assayed as descriid in NEN’s PG RIA kit. Briefly, 100 ~1 duplicates of samples and standards were transferred to 4 ml polyethylene tubes. ?- 3 G (100 ~1) was added to each tube and vortexed. PGl$ antibody (100 ~1) was added to each tube (except the bl 2k and tubes to identify the specific activity of the radiolabeled PGEJ and vortexed. The samples were incubated overnight in a refrigerator. One ml of precipitating reagent (polyethylene glycol mw 6,000) was added to each tube (except tubes for specific activity) and vortexed. The samples were incubated for another 20-u) min on ice, then centrifuged at 2500 x g for 30 min in a chilled swinging bucket centrifuge. The supernate was discarded and the radioactivity of the pellet was measured by a gamma counter. The PG in each tube was calculated from the standard curve generated. Each data point represents the average of 2 uplicate assays.
RESULTS
AND DISCUSSION
For some time it has been known that retinoic acid can alter the ‘=I-EGF binding capacity in certain cells (20), although, it has been difficult to correlate the alterations in binding capacity with any physiologic actions of EGF. Komura ti & (21) associated retinoic acid-induced increase in EGF-stimulated thymidine incorporation with increased ‘*‘I-EGF binding, yet other investigators have not been able to duplicate this observation in other cell types (22). The effect of retinoids on other EGF-induced responses, such as prostanoid secretion, has yet to be determined. A fetal rat lung cell line (FRL) was chosen to study the effects of retinoic acid on EGF responses, because both agents appear to 1516
BIOCHEMICAL
Vol. 162, No. 3, 1989
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PH EGF
+
Agent Untreated Retinoic Acid
5 7
+ 7.62 7.40
9 45
7.28 6.78
Cells were incubated for 24 hrs in the presence or absence of retinoic acid (5 PM). The cells were rinsed and incubated for another 24 hrs in serum-free media in the absence or presence of EGF (25 rig/ml) and retinoic acid. The media was then collected, the pH measured and duplicate 100 ~1 aliquot assayed for PGE, content. The PGE, values represent the average of the duplicates.
influence lung maturation (2-7). Furthermore, retinoic acid has been shown, in these cells, to stimulate a 3-fold increase in the expression of EGF receptors (1). To determine
the effects of retinoic
acid and EGF on prostaglandin
secretion,
confluent FRL cells were preincubated for 24 hrs in the presence or absence of retinoic acid (5 PM). One-half of each group was then stimulated with EGF (25 rig/ml) for 24 hrs and the medium from each sample was assayed, by a radioimmuno
assay (RIA), for PGS.
The results (Table 1) indicate that without retinoic acid preincubation, FRL cells secreted only a slightly higher level (approximately 80%) of PGE, in response to EGF addition. However, EGF addition interacts synergistically with retinoic acid pretreatment
of FRL cells
inducing a 6.4-fold increase in PGE, secretion. Interestingly,
a decrease in media pH that paralleled the level of PGE, secretion was
observed in cultures incubated in the absence or presence of retinoic acid and/or EGF (Table 1). Since prostaglandins are often released in response to injury, it could be argued that the acidic conditions
signaled the injury response producing prostaglandins.
This
notion, however, does not seem likely because acidic media (pH 6.9), altered by .l % HCl (lM), did not increase PGE, secretion when incubated with untreated FRL cells (data not shown).
These results suggest that although altered pH may be associated with PGS
release, it is not the cause. In retinoic acid treated FRL cells, increased levels of PGE, can be detected in the medium 2 hrs after EGF addition, nonetheless, the differential influence of EGF on control and retinoic acid treated cells becomes more pronounced as PGE, levels accumulate over a 48 hr time course (Figure 1). To determine whether the increase in PGE, secretion was associated with increases in EGF binding capacity, parallel cultures of cells treated with increasing concentrations of 1517
Vol. 162, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
120
I
.a* ’ unboatoo -O- +EGF -*a-. + R&ii + Reb +
, A& Acid & EGF
/’
/
,’
/
/
/’
1”
/ /’
<
0
........_..*............-......... .eo *..................._....................
I
I
I
12
24
36
..
. ... ...... ......’
‘I”nneAfter EGF AdUtii (hs)
Frc;uaEl. ~~~C-OFEGF--~-OPPG~~-ACID ‘lkxnur Fl2.L m Cells were pretreated for 24 hrs in the presence or absence of retinoic acid (5 PM). The cells were washed twice and refed with serum-free media (retinoic acid wasalso re-added to appropriate cultures) and incubated in the presence or absence of EGF (25 rig/ml). At the indicated times the media from specified samples wascollected and frozen. After all of the samples were collected the media was thawed and duplicate 100 ~1aliquot were assayedfor the presence of PGE;.
retinoic acid for 24 hrs were analyzed for either PGE, secretion or ‘=I-EGF binding capacity. The results demonstrate a retinoic acid concentration-dependent correlation between retinoid induced increases in”= I-EGF binding capacity and EGF-stimulated PGE, secretion (Figure 2). Retinoic acid concentrations of l-10 PM produced maximal increases in both 125I-EGF binding and PGE, secretion. Similarly, in cells treated with retinoic acid and then incubated with increasing concentrations of EGF or ‘%EGF, respectively, there were similar increases in the level of PGE, secreted or radiolabeled ligand binding (Figure 3). Maximal potentiation of PGE, secretion by EGF required concentrations of the growth factor sufficient to achieve near saturation of binding. These data demonstrate a correlation between the extent of EGF receptor occupancy and this EGF-induced response. Collectively, the results of both dose-response experiments suggest a relationship between the retinoic acid-induced increase in EGF receptor numbers and EGF responsiveness, although the influence of retinoic acid on other aspects of cell metabolism 1518
Vol. 162, No. 3, 1989
BIOCHEMICAL
Retinoic
0.0
aAcId
L\\
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
bnc+dMr)
I
c
I
10 -09
10
-
10
I
1
10 a
-07
10
-05
10 -a
Retinoic Acid (moles/liter)
FIGURE 2.
ACID CON -TION ON EGF-STIMULMED ml!+s REIUSEL Cellswere incubatedwith various concentrationsof retinoic acid for 24 hrs. The cell were then washedand refed with serum-freemedia,EGF (25 rig/ml) and the appropriate concentrationof retinoic acid. After incubation for another 24 hrs, the media was collected and assayedfor the presenceof PGQ. (Inset) Effect of various retinoic acid concentrationson ‘=I-EGF binding. 125
Gi 4 rn = 8 0
TEE Eppecr OF R~INOIC
I
28,
retinoic
100
0 75 F5 0. -0 g50 iirn
9
0
20
40
60 so EGF hglml)
acid treated
loo
120
Retinoic Acid
f N 25
0
10
20
30
40
50
EGF (rig/ml)
FIGDRE 3.
PGE, !hCREIlON: EGF DOSE-REsmNsrr.~~J-IWEM &IINOIC Acm m ZEUS. Cellswere incubatedwith retinoic acid (5 PM) for 24 hrs. The cell were then washedand refed with serum-freemedia, EGF at various concentrationsand retinoic acid (in the appropriatecultures). After incubationfor another 24 hrs, the media was collected and assayedfor the presenceof PGQ. (Inset) ‘?-EGF binding as a function of EGF concentration.
%EA’lICD
1519
Vol.
162,
No.
3, 1989
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
that could potentiate the effects of EGF can not be ruled out. Irrespective of the mechanism, the observation that retinoic acid and EGF interact synergistically to enhance PGE, release is significant.
Since PGE, has been shown to enhance pulmonary surfactant
secretion (14 & 15) these data may indicate a mechanism by which retinoic acid and EGF participate
in lung function.
ACKNOWLEDGMENTS We thank Kelly Adair for excellent technical assistance. This research was supported by grants HI-14214 from the NIH and a grant from the Walter E. Macpherson Society.
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