Prostaglandins Leukotrienes and Essential Fatty Acids (1991) 44. 57-59 0 Longman Group UK Ltd 1991
Epidermal Growth Factor and the Regulation of Amnion Prostaglandin Biosynthesis M. D. Mitchell, S. S. Edwin, R. Lepera and S. Lundin-Schiller Department of Obstetrics and Gynecology, University of Utah Medical Center, 50 N. Medical Drive, Salt Lake City, Utah 84132, USA (Reprint requests to MDM)
The production of prostagiandins by amnion is a key factor in the mechanism of human parturition yet the regulation of prostagiandin biosynthesis in amnion is poorly understood. Hence, we have investigated the regulation of epidermal growth factor (EGF) stimulation of prostaglandin biosynthesis in human amnion cells. This stimulatory action is inhibited by cycloheximide or actinomycin D at high concentrations, but enhanced at much lower concentrations of these protein synthesis inhibitors. An amnion-produced prostagiandin inhibitor or immediate early gene action may explain these effects. Pretreatment with phorbol esters (inhibition of protein kinase C activity) reduces basal prostaglandht production and attenuates the stimulatory action of EGF on prostaglandin biosynthesis. Hence, amnion prostaglandin biosynthesis is dependent partly on protein kinase C activity.
ABSTRACT.
INTRODUCTION
EXPERIMENTAL PROCEDURES Cell culture
It has become apparent
that prostaglandin production by amnion plays a significant part in the mechanism(s) of human parturition (1). Nevertheless, there is a paucity of information concerning the regulation of prostaglandin biosynthesis in amnion. Epidermal growth factor (EGF) may have a significant role in the process of parturition since its concentration in amniotic fluid is elevated during labor (2) and it has important actions on lung maturation (3) and adrenal function (4). Moreover, EGF stimulates amnion cell prostaglandin production (5) in part by an action on prostaglandin H2 synthase (6). An action of EGF to induce release of substrate in amnion has been proposed (5) and would be consistent with findings in other cell types including epidermoid carcinoma cells (7). Thus, we decided to evaluate the actions of protein synthesis inhibitors on EGF stimulation of amnion prostaglandin biosynthesis. Furthermore, since we (8) and others (9, 10) have described the stimulation of amnion prostaglandin biosynthesis by phorbol esters, we have further examined the possible role of protein kinase C in this process.
Placentae were obtained at nine elective repeat cesarean sections and amnion cells isolated as described previously (5, 11). Briefly, amnion tissue was dissected from the chorion laeve and dispersed by incubation in trypsin (1.2% in Dulbecco’s Modified Eagles Medium) for 2 h at 37°C. Amnion cells were plated in Ham’s F12:Dulbecco’s Modified Eagles Medium (1: 1) [Ham’s F12:DME] plus 10% fetal calf serum [FCS] at a density of 300 000 cells/well in 24-16 mm well plates. Cells were maintained at 37°C in an atmosphere of 5% CO2 in air. When the cultures were confluent experiments were conducted with each experimental condition in quadruplicate. Experimental conditions were prepared in Ham’s F12:DME plus 10% FCS. Trypsin was obtained from Difco (Detroit, MI). Culture media and FCS were obtained from Irvine Scientific (Santa Ana, CA). Tissue culture plates were obtained from Costar (Cambridge, MA). Incubations incubations were conducted for 16 h (or 24 h for pretreatment) in the presence and absence of phorbol 1Zmyristate 13-acetate (PMA), EGF (both Calbiochem, La Jolla, CA), 4 (Yphorbol-didecanoate (PDD), cycloheximide and actinomycin D (all from
Date received 15 February 1991 Date accepted 25 April 1991 51
58
Prostaglandins Leukotrienes
and Essential Fatty Acids
Sigma, St. Louis, MO). In experiments in which cells were pretreated with PMA (10e7M) or PDD (10e7M) for 24 h, the pretreatment was removed and the cells were rinsed twice with fresh media prior to incubation with other substances. Previous studies (12, 13) have indicated that this concentration of PMA for 24 h should substantially inhibit protein kinase C activity. Assays
Prostaglandin E2 (PGEQ) is the major prostaglandin synthesized by amnion and was measured directly in the culture media as described previously (5) using an antiserum from Advanced Magnetics Inc (Cambridge, MA). Cellular protein was determined by the method of Lowry et al (14). Statistical differences were assessed using Students’ t-test.
1 CCNltrOl
PMA
EGF
Fig. 2 The effects of media (0) or PMA (lo-‘M) (@) pretreatment (24 h) of human amnion cells on basal and PMA or EGF-stimulated prostaglandin production (mean f SEM, n = 6). The results presented are from means of six individual experiments. For pertinent statistical differences see text.
RESULTS
The stimulatory action of EGF on amnion prostaglandin biosynthesis has a requirement for new protein synthesis as demonstrated by attenuation or abolition of this action by cycloheximide or actinomycin D at 10 pg/rnl (Fig. 1). The degree of stimulation was reduced by approximately 65% (p
Epidermal Growth Factor and the Regulation of Amnion Prostaglandin Biosynthesis M. D. Mitchell, S. S. Edwin, R. Lepera and S. Lundin-Schiller Department of Obstetrics and Gynecology, University of Utah Medical Center, 50 N. Medical Drive, Salt Lake City, Utah 84132, USA (Reprint requests to MDM)
The production of prostagiandins by amnion is a key factor in the mechanism of human parturition yet the regulation of prostagiandin biosynthesis in amnion is poorly understood. Hence, we have investigated the regulation of epidermal growth factor (EGF) stimulation of prostaglandin biosynthesis in human amnion cells. This stimulatory action is inhibited by cycloheximide or actinomycin D at high concentrations, but enhanced at much lower concentrations of these protein synthesis inhibitors. An amnion-produced prostagiandin inhibitor or immediate early gene action may explain these effects. Pretreatment with phorbol esters (inhibition of protein kinase C activity) reduces basal prostaglandht production and attenuates the stimulatory action of EGF on prostaglandin biosynthesis. Hence, amnion prostaglandin biosynthesis is dependent partly on protein kinase C activity.
ABSTRACT.
INTRODUCTION
EXPERIMENTAL PROCEDURES Cell culture
It has become apparent
that prostaglandin production by amnion plays a significant part in the mechanism(s) of human parturition (1). Nevertheless, there is a paucity of information concerning the regulation of prostaglandin biosynthesis in amnion. Epidermal growth factor (EGF) may have a significant role in the process of parturition since its concentration in amniotic fluid is elevated during labor (2) and it has important actions on lung maturation (3) and adrenal function (4). Moreover, EGF stimulates amnion cell prostaglandin production (5) in part by an action on prostaglandin H2 synthase (6). An action of EGF to induce release of substrate in amnion has been proposed (5) and would be consistent with findings in other cell types including epidermoid carcinoma cells (7). Thus, we decided to evaluate the actions of protein synthesis inhibitors on EGF stimulation of amnion prostaglandin biosynthesis. Furthermore, since we (8) and others (9, 10) have described the stimulation of amnion prostaglandin biosynthesis by phorbol esters, we have further examined the possible role of protein kinase C in this process.
Placentae were obtained at nine elective repeat cesarean sections and amnion cells isolated as described previously (5, 11). Briefly, amnion tissue was dissected from the chorion laeve and dispersed by incubation in trypsin (1.2% in Dulbecco’s Modified Eagles Medium) for 2 h at 37°C. Amnion cells were plated in Ham’s F12:Dulbecco’s Modified Eagles Medium (1: 1) [Ham’s F12:DME] plus 10% fetal calf serum [FCS] at a density of 300 000 cells/well in 24-16 mm well plates. Cells were maintained at 37°C in an atmosphere of 5% CO2 in air. When the cultures were confluent experiments were conducted with each experimental condition in quadruplicate. Experimental conditions were prepared in Ham’s F12:DME plus 10% FCS. Trypsin was obtained from Difco (Detroit, MI). Culture media and FCS were obtained from Irvine Scientific (Santa Ana, CA). Tissue culture plates were obtained from Costar (Cambridge, MA). Incubations incubations were conducted for 16 h (or 24 h for pretreatment) in the presence and absence of phorbol 1Zmyristate 13-acetate (PMA), EGF (both Calbiochem, La Jolla, CA), 4 (Yphorbol-didecanoate (PDD), cycloheximide and actinomycin D (all from
Date received 15 February 1991 Date accepted 25 April 1991 51
58
Prostaglandins Leukotrienes
and Essential Fatty Acids
Sigma, St. Louis, MO). In experiments in which cells were pretreated with PMA (10e7M) or PDD (10e7M) for 24 h, the pretreatment was removed and the cells were rinsed twice with fresh media prior to incubation with other substances. Previous studies (12, 13) have indicated that this concentration of PMA for 24 h should substantially inhibit protein kinase C activity. Assays
Prostaglandin E2 (PGEQ) is the major prostaglandin synthesized by amnion and was measured directly in the culture media as described previously (5) using an antiserum from Advanced Magnetics Inc (Cambridge, MA). Cellular protein was determined by the method of Lowry et al (14). Statistical differences were assessed using Students’ t-test.
1 CCNltrOl
PMA
EGF
Fig. 2 The effects of media (0) or PMA (lo-‘M) (@) pretreatment (24 h) of human amnion cells on basal and PMA or EGF-stimulated prostaglandin production (mean f SEM, n = 6). The results presented are from means of six individual experiments. For pertinent statistical differences see text.
RESULTS
The stimulatory action of EGF on amnion prostaglandin biosynthesis has a requirement for new protein synthesis as demonstrated by attenuation or abolition of this action by cycloheximide or actinomycin D at 10 pg/rnl (Fig. 1). The degree of stimulation was reduced by approximately 65% (p
