0013-7227/91/1285-2651$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 5 Printed in U.S.A.
Epidermal growth factor and phorbol esters reduce the levels of the cognate mRNA for the LH/CG receptor Haiyun Wangi, Deborah L. Segaloffi, and Mario Ascoli§ IDepartment of Physiology and Biophysics and §Department of Pharmacology, The University of Iowa College of Medicine,Iowa City, IA 52242-1109 The LH/CG receptor of MA-10 Leydig tumor cells can be down-regulated by LH/CG, mouse epidermal growth factor (mEGF), cAMP analogues and phorbol esters. In a recent publication we showed that the LH/CG-induced down regulation of the LH/CG receptor is primarily due to an increase in the rate of degradation of the receptor, and occurs with little or no change in the levels of LH/CG receptor mRNA. Although relatively unimportant for the LH/CG-induced down-regulation of the LH/CG receptor, we also found that LH/CG can in fact elicit a reduction in LH/CG receptor mRNA and that this reduction is mediated by cAMP. These findings raised the possibility that the ability of other compounds to down-regulate the LH/CG receptor may be accompanied by decreases in LH/CG receptor mRNA. In an attempt to answer this question, we examined the possibility that the mEGF- or phorbol ester-induced down regulation of the LH/CG receptor is due to changes in LH/CG receptor mRNA. The experiments presented herein show that this is indeed the case. Introduction
Materials and Methods
Previous studies from this and other laboratories have shown that the LH/CG receptor of Leydig tumor cells can be down regulated by LH/CG (1-4), mEGF (1,5) and certain second messengers analogues such as 8-Br-cAMP and phorbol esters (3,4,6).
Standard Experimental Protocol. The origin and handling of MA-10 and MA-10(K3) cells have been described (4.11). The methods used to measure the binding of I-hCG, to isolate cytoplasraic RNA, and to measure the levels of LH/CG receptor mRNA by solution hybridization have all been described in detail (4). Hormones and Supplies. Purified hCG (CR-125, 11,900 U/mg) was obtained from the National Hormone and Pituitary Program. Mouse EGF was generously provided by Dr. Graham Carpenter (Vanderbilt University). All other materials were obtained as described recently (4).
Using indirect methods, we had previously concluded that the LH/CG-induced and mEGF-induced down-regulation of the LH/CG receptor occurred by different mechanisms (1). While LH/CG were found to increase the rate of receptor degradation, mEGF appeared to decrease the rate of receptor synthesis. Because of the recent availability of cDNA probes for the LH/CG receptor (7,8), it is now possible to more directly examine the molecular mechanisms responsible for its regulation. Indeed, in a recent publication we used these tools to show that LH/CG can substantially reduce LH/CG receptors without a parallel reduction in receptor mRNA (4). This finding is consistent with our previous conclusion that the principal mechanism involved in the LH/CG-induced down-regulation of the LH/CG receptor involves an increase in the rate of receptor degradation resulting from the increased accumulation of the hormone-receptor complex in the lysosomes that occurs during the receptor-mediated endocytosis of LH/CG (1,9,10). We also showed that a reduction in LH/CG receptor mRNA occurs upon prolonged incubation of MA-10 cells with LH/CG, and that this reduction is mediated by cAMP (4). Therefore, the previously documented ability of cAMP analogues to down-regulate LH/CG receptors in Leydig tumor cells (3) can now be entirely explained by their ability to reduce the receptor mRNA (4).
Results and Discussion In order to determine if the mEGF- or phorbol 12-myristate 13-acetate (PMA)-induced downregulation of the LH/CG receptor is accompanied by changes in the levels of LH/CG receptor mRNA, we incubated MA-10 cells with a maximally effective concentration of either compound, and then measured the binding of I-hCG and the levels of receptor mRNA at different times after addition. The levels of receptor mRNA were measured by solution hybridization using a method recently developed by us (4). It is important to note that, like other gonadal tissues, MA-10 cells express several LH/CG receptor mRNA transcripts. These transcripts change in a coordinate fahsion when the overall levels of LH/CG receptor mRNA are reduced (4). The results presented in Fig 1 show that, as expected (1,5,6)^ addition of mEGF or PMA leads to a reduction in I-hCG binding. Upon addition of either compound there is a lag of about 6 h before a decrease in binding is detected. In fact, during this time it is not uncommon to detect a small (1020%) increase in binding. At subsequent times the binding declines slowly and reaches a new steady state (corresponding to 20-40% of the initial levels) within 40 h. The time course and magnitude of this down-regulation of I-hCG binding are very similar with these two compounds. The results presented in Fig 1 also show, for the first time, that mEGF and PMA are capable of reducing the levels of LH/CG receptor mRNA. This decrease in mRNA seems to be sufficient to account for the reduction in LH/CG receptors, because the time course of the reduction in mRNA precedes that of the reduction in binding activity and because at
Since the time courses involved in the downregulation of the LH/CG receptor by mEGF and phorbol esters are similar to each other and to that observed with cAMP analogues (1,3-6), we speculated that they down regulate LH/CG receptors by decreasing receptor synthesis. We now present studies designed to investigate this possibility.
Received in Iowa City:
February 19, 1991
2651
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 03:32 For personal use only. No other uses without permission. . All rights reserved.
2652
RAPID COMMUNICATIONS
steady state the magnitude of the decrease in binding and mRNA are similar (or identical). In other experiments (not presented) we showed that phorbol 13,20-diacetate, aiv inactive phorbol ester, was unable to decrease I-hCG binding and LH/CG receptor mRNA in MA-10 cells.
Endo • 1991 Voll28«No5
the possibility that the observed effects of mEGF are mediated by activation of protein kinase C. This is important because mEGF activates phospholipase Cy in some cell types (13,14). It should be noted, however, that the activation of this pathway by mEGF is not a universal phenomenon (15). In fact, we have shown that mEGF does not elicit an increase in inositpl phosphates, diacylglycerol or intracellular Ca in MA-10 cells (ref 16 and unpublished observations). Thus, it is unlikely that the effects of mEGF reported here are mediated by the activation of protein kinase C.
100
mEGF
PMA
mEGF
PMA
Figure 2. Effects of mEGF and PMA on the levels of
hours Figure 1.
Effects of mEGF and PMA on the levels of
125
I-hCG binding and LH/CG receptor mRNA. MA-10 cells were incubated with 5 ng/ral mEGF or 200 nM PMA as indicated. Each point represents the average (± SEM) of 4 different determinations (2 experiments with duplicate dishes in each experiment). In a recent publication we reported that 8-BrcAMP reduces the levels of I-hCG binding and LH/CG receptor mRNA and that this reduction requires an active cAMP-dependent protein kinase (4). In order to test if the same requirement is needed for the expression of the mEGF and PMA effects, we compared the ability of these compounds to reduce I-hCG binding and LH/CG receptor mRNA in the wild-type MA-10 cells and in a subclone of MA-10 cells (designated MA-10(K3)) that express a cAMPresistant phenotype (12). The results presented in Fig 2 show that mEGF or PMA reduce LH/CG receptors and LH/CG receptor mRNA to about the same extent in both cell types. These results show that the reported effects of mEGF or PMA do not require an active cAMP-dependent protein kinase. Since PMA faithfully mimics the effects of mEGF on I~hCG binding and LH/CG receptor mRNA, one must consider
125 I-hCG binding and LH/CG receptor mRNA in MA-10 and MA-10(K3) cells. MA-10 and MA-10(K3) cells were preincubated in growth medium containing 100 mM ZnSO^ for 8 h to induce the cAMP resistant phenotype (see ref 12). PMA (200 nM) or mEGF (5 ng/ml) were then added as indicated and the incubation was continued for an additional 40 h. Each point represents the average (± SEM) of 4-6 different determinations (2-3 experiments with duplicate dishes in each experiment).
Last, it should be noted that the ability of mEGF and phorbol ester to down-regulate the LH/CG receptor can also be observed in granulosa cells (17-19). Based on the results presented here we would predict that these compounds also decrease the LH/CG receptor mRNA in granulosa cells. References 1.
2.
3.
Lloyd CE, Ascoli M 1983 On the mechanisms involved in the regulation of the cell surface receptors for human chorlogonado-" tropin and mouse epidermal growth factor in cultured Leydig tumor cells. J. Cell Biol. 96: 521-526 Rebois RV, Fishman PH 1983 Deglycosylated human chorionic gonadotropin. An antagonist to desensitization and down-regulation of the gonadotropin receptor-adenylate cyclase system. J. Biol. Chem. 258: 12775-12778 Rebois RV, Fishman PH 1984 Down-regulation of gonadotropin receptors in a murine Leydig tumor cell line. J. Biol. Chem. 259: 30963101
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 03:32 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Wang H, Segaloff DL, Ascoli M 1991 Lutropin/choriogonadotropin down-regulates its receptor by both receptor mediated endocytosis and a cAMP-dependent reduction in receptor mRNA. J. Biol. Chem. 266: 780-785 Ascoli M 1981 Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor. J. Biol. Chem. 256: 179-183 Rebois RV, Patel J 1985 Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line. J. Biol. Chem. 260: 8026-8031 McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G proteincoupled receptor family. Science 245: 494-499 Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Gamier J, Milgrom E 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmerabrane domain. Science 245: 525-528 Ascoli M 1982 Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of the hormone subunits. J. Biol. Chem. 257: 13306-13311 Ascoli M 1984 Lysosomal accumulation of the hormone-receptor complex during receptormediated endocytosis of human choriogonadotropin. J. Cell Biol. 99: 12421250 Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108: 88-95 Wang H, Ascoli M 1990 Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA10 cells with a mutant gene of the type I regulatory subunit of the cAMP-dependent protein kinase. Mol. Endocrinol. 4: 80-90 Wahl MI, Daniel TO, Carpenter G 1988 Antiphosphotyrosine recovery of phospholipase C activity after EGF treatment of A-431 cells. Science 241: 968-970 Meisenhelder J, Suh P-G, Rhee S-G, Hunter T 1989 Phospholipase Cy is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 57: 1109-1122 Olashaw NE, Pledger WJ 1988 Epidermal growth factor stimulates formation of inositol phosphates in BALB/c/3T3 cells pretreated with cholera toxin and isobutylxanthine. J. Biol. Chera. 263: 1111-1114 Ascoli M, Pignataro OP, Segaloff DL 1989 The inositol phosphate/diacylglycerol pathway in MA-10 Leydig tumor cells. Activation by arginine vasopressin and lack of effect of epidermal growth factor and human choriogonadotropin. J. Biol. Chem. 264: 66746681 Kasson BG, Conn PM, Hsueh AJW 1985 Inhibition of granulosa cell differentiation by dioctanoylglycerol, novel activator of protein kinase C. Mol. Cell. Endocrinol. 42: 29-37
18.
19.
2653
Mondschein JS, Schomberg DW 1981 Growth factors modulate gonadotropin receptor induction in granulosa cell cultures. Science 211: 1179-1180 Knecht M, Catt KJ 1983 Modulation of cAMPmediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet derived growth factor. J. Biol. Chem. 258: 2789-2794 Acknowledgments
We wish to thank Bruce D'Souza for expert assistance with the cell culture. This work was supported by grants from the National Institutes of Health: HD-22196 (to DLS), CA-40629 (to M A ) , and DK-25295 (Diabetes and Endocrinology Research Center of the University of Iowa) and by the Roy J. Carver Charitable Trust (to M A ) . Address correspondence to Dr. Mario Ascoli, Dept. of Pharmacology, University of Iowa College of Medicine, Iowa City, IA 52242-1109.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 03:32 For personal use only. No other uses without permission. . All rights reserved.
Vol. 128, No. 5 Printed in U.S.A.
Epidermal growth factor and phorbol esters reduce the levels of the cognate mRNA for the LH/CG receptor Haiyun Wangi, Deborah L. Segaloffi, and Mario Ascoli§ IDepartment of Physiology and Biophysics and §Department of Pharmacology, The University of Iowa College of Medicine,Iowa City, IA 52242-1109 The LH/CG receptor of MA-10 Leydig tumor cells can be down-regulated by LH/CG, mouse epidermal growth factor (mEGF), cAMP analogues and phorbol esters. In a recent publication we showed that the LH/CG-induced down regulation of the LH/CG receptor is primarily due to an increase in the rate of degradation of the receptor, and occurs with little or no change in the levels of LH/CG receptor mRNA. Although relatively unimportant for the LH/CG-induced down-regulation of the LH/CG receptor, we also found that LH/CG can in fact elicit a reduction in LH/CG receptor mRNA and that this reduction is mediated by cAMP. These findings raised the possibility that the ability of other compounds to down-regulate the LH/CG receptor may be accompanied by decreases in LH/CG receptor mRNA. In an attempt to answer this question, we examined the possibility that the mEGF- or phorbol ester-induced down regulation of the LH/CG receptor is due to changes in LH/CG receptor mRNA. The experiments presented herein show that this is indeed the case. Introduction
Materials and Methods
Previous studies from this and other laboratories have shown that the LH/CG receptor of Leydig tumor cells can be down regulated by LH/CG (1-4), mEGF (1,5) and certain second messengers analogues such as 8-Br-cAMP and phorbol esters (3,4,6).
Standard Experimental Protocol. The origin and handling of MA-10 and MA-10(K3) cells have been described (4.11). The methods used to measure the binding of I-hCG, to isolate cytoplasraic RNA, and to measure the levels of LH/CG receptor mRNA by solution hybridization have all been described in detail (4). Hormones and Supplies. Purified hCG (CR-125, 11,900 U/mg) was obtained from the National Hormone and Pituitary Program. Mouse EGF was generously provided by Dr. Graham Carpenter (Vanderbilt University). All other materials were obtained as described recently (4).
Using indirect methods, we had previously concluded that the LH/CG-induced and mEGF-induced down-regulation of the LH/CG receptor occurred by different mechanisms (1). While LH/CG were found to increase the rate of receptor degradation, mEGF appeared to decrease the rate of receptor synthesis. Because of the recent availability of cDNA probes for the LH/CG receptor (7,8), it is now possible to more directly examine the molecular mechanisms responsible for its regulation. Indeed, in a recent publication we used these tools to show that LH/CG can substantially reduce LH/CG receptors without a parallel reduction in receptor mRNA (4). This finding is consistent with our previous conclusion that the principal mechanism involved in the LH/CG-induced down-regulation of the LH/CG receptor involves an increase in the rate of receptor degradation resulting from the increased accumulation of the hormone-receptor complex in the lysosomes that occurs during the receptor-mediated endocytosis of LH/CG (1,9,10). We also showed that a reduction in LH/CG receptor mRNA occurs upon prolonged incubation of MA-10 cells with LH/CG, and that this reduction is mediated by cAMP (4). Therefore, the previously documented ability of cAMP analogues to down-regulate LH/CG receptors in Leydig tumor cells (3) can now be entirely explained by their ability to reduce the receptor mRNA (4).
Results and Discussion In order to determine if the mEGF- or phorbol 12-myristate 13-acetate (PMA)-induced downregulation of the LH/CG receptor is accompanied by changes in the levels of LH/CG receptor mRNA, we incubated MA-10 cells with a maximally effective concentration of either compound, and then measured the binding of I-hCG and the levels of receptor mRNA at different times after addition. The levels of receptor mRNA were measured by solution hybridization using a method recently developed by us (4). It is important to note that, like other gonadal tissues, MA-10 cells express several LH/CG receptor mRNA transcripts. These transcripts change in a coordinate fahsion when the overall levels of LH/CG receptor mRNA are reduced (4). The results presented in Fig 1 show that, as expected (1,5,6)^ addition of mEGF or PMA leads to a reduction in I-hCG binding. Upon addition of either compound there is a lag of about 6 h before a decrease in binding is detected. In fact, during this time it is not uncommon to detect a small (1020%) increase in binding. At subsequent times the binding declines slowly and reaches a new steady state (corresponding to 20-40% of the initial levels) within 40 h. The time course and magnitude of this down-regulation of I-hCG binding are very similar with these two compounds. The results presented in Fig 1 also show, for the first time, that mEGF and PMA are capable of reducing the levels of LH/CG receptor mRNA. This decrease in mRNA seems to be sufficient to account for the reduction in LH/CG receptors, because the time course of the reduction in mRNA precedes that of the reduction in binding activity and because at
Since the time courses involved in the downregulation of the LH/CG receptor by mEGF and phorbol esters are similar to each other and to that observed with cAMP analogues (1,3-6), we speculated that they down regulate LH/CG receptors by decreasing receptor synthesis. We now present studies designed to investigate this possibility.
Received in Iowa City:
February 19, 1991
2651
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 03:32 For personal use only. No other uses without permission. . All rights reserved.
2652
RAPID COMMUNICATIONS
steady state the magnitude of the decrease in binding and mRNA are similar (or identical). In other experiments (not presented) we showed that phorbol 13,20-diacetate, aiv inactive phorbol ester, was unable to decrease I-hCG binding and LH/CG receptor mRNA in MA-10 cells.
Endo • 1991 Voll28«No5
the possibility that the observed effects of mEGF are mediated by activation of protein kinase C. This is important because mEGF activates phospholipase Cy in some cell types (13,14). It should be noted, however, that the activation of this pathway by mEGF is not a universal phenomenon (15). In fact, we have shown that mEGF does not elicit an increase in inositpl phosphates, diacylglycerol or intracellular Ca in MA-10 cells (ref 16 and unpublished observations). Thus, it is unlikely that the effects of mEGF reported here are mediated by the activation of protein kinase C.
100
mEGF
PMA
mEGF
PMA
Figure 2. Effects of mEGF and PMA on the levels of
hours Figure 1.
Effects of mEGF and PMA on the levels of
125
I-hCG binding and LH/CG receptor mRNA. MA-10 cells were incubated with 5 ng/ral mEGF or 200 nM PMA as indicated. Each point represents the average (± SEM) of 4 different determinations (2 experiments with duplicate dishes in each experiment). In a recent publication we reported that 8-BrcAMP reduces the levels of I-hCG binding and LH/CG receptor mRNA and that this reduction requires an active cAMP-dependent protein kinase (4). In order to test if the same requirement is needed for the expression of the mEGF and PMA effects, we compared the ability of these compounds to reduce I-hCG binding and LH/CG receptor mRNA in the wild-type MA-10 cells and in a subclone of MA-10 cells (designated MA-10(K3)) that express a cAMPresistant phenotype (12). The results presented in Fig 2 show that mEGF or PMA reduce LH/CG receptors and LH/CG receptor mRNA to about the same extent in both cell types. These results show that the reported effects of mEGF or PMA do not require an active cAMP-dependent protein kinase. Since PMA faithfully mimics the effects of mEGF on I~hCG binding and LH/CG receptor mRNA, one must consider
125 I-hCG binding and LH/CG receptor mRNA in MA-10 and MA-10(K3) cells. MA-10 and MA-10(K3) cells were preincubated in growth medium containing 100 mM ZnSO^ for 8 h to induce the cAMP resistant phenotype (see ref 12). PMA (200 nM) or mEGF (5 ng/ml) were then added as indicated and the incubation was continued for an additional 40 h. Each point represents the average (± SEM) of 4-6 different determinations (2-3 experiments with duplicate dishes in each experiment).
Last, it should be noted that the ability of mEGF and phorbol ester to down-regulate the LH/CG receptor can also be observed in granulosa cells (17-19). Based on the results presented here we would predict that these compounds also decrease the LH/CG receptor mRNA in granulosa cells. References 1.
2.
3.
Lloyd CE, Ascoli M 1983 On the mechanisms involved in the regulation of the cell surface receptors for human chorlogonado-" tropin and mouse epidermal growth factor in cultured Leydig tumor cells. J. Cell Biol. 96: 521-526 Rebois RV, Fishman PH 1983 Deglycosylated human chorionic gonadotropin. An antagonist to desensitization and down-regulation of the gonadotropin receptor-adenylate cyclase system. J. Biol. Chem. 258: 12775-12778 Rebois RV, Fishman PH 1984 Down-regulation of gonadotropin receptors in a murine Leydig tumor cell line. J. Biol. Chem. 259: 30963101
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 03:32 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Wang H, Segaloff DL, Ascoli M 1991 Lutropin/choriogonadotropin down-regulates its receptor by both receptor mediated endocytosis and a cAMP-dependent reduction in receptor mRNA. J. Biol. Chem. 266: 780-785 Ascoli M 1981 Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor. J. Biol. Chem. 256: 179-183 Rebois RV, Patel J 1985 Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line. J. Biol. Chem. 260: 8026-8031 McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G proteincoupled receptor family. Science 245: 494-499 Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Gamier J, Milgrom E 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmerabrane domain. Science 245: 525-528 Ascoli M 1982 Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of the hormone subunits. J. Biol. Chem. 257: 13306-13311 Ascoli M 1984 Lysosomal accumulation of the hormone-receptor complex during receptormediated endocytosis of human choriogonadotropin. J. Cell Biol. 99: 12421250 Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108: 88-95 Wang H, Ascoli M 1990 Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA10 cells with a mutant gene of the type I regulatory subunit of the cAMP-dependent protein kinase. Mol. Endocrinol. 4: 80-90 Wahl MI, Daniel TO, Carpenter G 1988 Antiphosphotyrosine recovery of phospholipase C activity after EGF treatment of A-431 cells. Science 241: 968-970 Meisenhelder J, Suh P-G, Rhee S-G, Hunter T 1989 Phospholipase Cy is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 57: 1109-1122 Olashaw NE, Pledger WJ 1988 Epidermal growth factor stimulates formation of inositol phosphates in BALB/c/3T3 cells pretreated with cholera toxin and isobutylxanthine. J. Biol. Chera. 263: 1111-1114 Ascoli M, Pignataro OP, Segaloff DL 1989 The inositol phosphate/diacylglycerol pathway in MA-10 Leydig tumor cells. Activation by arginine vasopressin and lack of effect of epidermal growth factor and human choriogonadotropin. J. Biol. Chem. 264: 66746681 Kasson BG, Conn PM, Hsueh AJW 1985 Inhibition of granulosa cell differentiation by dioctanoylglycerol, novel activator of protein kinase C. Mol. Cell. Endocrinol. 42: 29-37
18.
19.
2653
Mondschein JS, Schomberg DW 1981 Growth factors modulate gonadotropin receptor induction in granulosa cell cultures. Science 211: 1179-1180 Knecht M, Catt KJ 1983 Modulation of cAMPmediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet derived growth factor. J. Biol. Chem. 258: 2789-2794 Acknowledgments
We wish to thank Bruce D'Souza for expert assistance with the cell culture. This work was supported by grants from the National Institutes of Health: HD-22196 (to DLS), CA-40629 (to M A ) , and DK-25295 (Diabetes and Endocrinology Research Center of the University of Iowa) and by the Roy J. Carver Charitable Trust (to M A ) . Address correspondence to Dr. Mario Ascoli, Dept. of Pharmacology, University of Iowa College of Medicine, Iowa City, IA 52242-1109.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 03:32 For personal use only. No other uses without permission. . All rights reserved.
