0021-972X/91/7206-1261$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 72, No. 6 Printed in U.S.A.
The Effects of Activin on Follicle-Stimulating Hormone Secretion and Biosynthesis in Human Glycoprotein Hormone-Producing Pituitary Adenomas* JOSEPH M. ALEXANDER, J. LARRY JAMESON, HELEN A. BIKKAL, RALPH H. SCHWALL, AND ANNE KLIBANSKI Neuroendocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114; and the Department of Developmental Biology, Genentech, Inc. (R.H.S.), South San Francisco, California 94080
ABSTRACT. The effects of activin on pituitary FSH biosynthesis have been previously characterized using primary rat pituitary cultures; however, little is known of the effects of activin on FSH biosynthesis and secretion in human pituitary tissue. Production of intact glycoprotein hormones and free subunits is increasingly recognized in pituitary tumors; however, the regulation of gonadotropins in such tumors has not been addressed. We have investigated the effects of human recombinant activin on glycoprotein hormone biosynthesis and secretion in primary cultures of 12 human glycoprotein hormone-producing pituitary adenomas and compared this with the effects of activin in normal rat anterior pituitary cells. In 33% of the human pituitary tumors studied, significant (P < 0.05) increases in FSH/3 secretion occurred in response to incubation with 20 ng/mL activin for 24 h (19-287% stimulation), without changes in the production of intact FSH. A Northern analysis performed on cells derived from one tumor indicated that FSH/3 mRNA
levels increased 350% after activin treatments; however, FSH secretion did not parallel the mRNA changes. None of the human glycoprotein hormone-producing tumors significantly increased FSH secretion in response to activin. To validate the biological activity of recombinant human activin-A and to confirm time and dose conditions for the human tumor cultures, we also examined its ability to stimulate FSH production in rat pituitary cultures. Activin (20 ng/mL) added to the culture medium significantly increased FSH secretion and steady state levels of FSHjS mRNA after 24 h. These data indicate that some glycoprotein hormone-producing pituitary tumors treated with purified activin have discordant responses of intact gonadotropins and free subunit responses. In contrast to responses in normal rat gonadotrophs, FSH/3 biosynthetic pathways may be uncoupled from intact FSH secretion in a subset of glycoprotein hormone-producing pituitary adenomas. (J Clin Endocrinol Metab 72: 1261-1267, 1991)
C
LINICALLY nonfunctioning pituitary tumors represent 25-30% of the diagnosed pituitary adenomas and often secrete intact glycoprotein hormones and/or their free subunits (1). Light and electron microscopy studies of these tumors show the presence of secretory granules in neoplastic pituitary tissue (2), and the majority of these tumors secrete FSH or free a- and FSH/3subunits (3-5). RNA analysis (6-9) and immunocytochemistry (10-12) also confirm the synthesis of intact gonadotropins or their subunits by a subset of clinically nonfunctioning pituitary adenomas. FSH production in these tumors has been well documented, but the biosynthetic mechanisms that regulate FSH production and secretion in neoplastic pituitary cells are poorly understood. There is increasing evidence that gonadal peptides play an important role in the regulation of normal pituiReceived September 27, 1990. Address requests for reprints to: Anne Klibanski, M.D., Neuroendocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. * This work was supported by NIDDK Grants DK-40947, DK-07028, and HD-23519.
tary FSH biosynthesis. Therefore, it is essential to characterize the responses of neoplastic pituitary tumors to peptides that have selective effects on FSH biosynthesis and secretion. Activins are polypeptides of gonadal origin which specifically stimulate FSH release by pituitary gonadotrophs (13). The activin polypeptides are dimers consisting of two inhibin /3-subunits (14, 15). The three forms of activin, activin-A (0A/3A), activin-B (/3B/3B), and activinAB (/3A/3B), have been shown to have similar biological activity. Activin-A and -AB have been purified from natural sources, while bioactive activin-A and B have been produced using recombinant DNA technology (16). Sequence analysis of cloned inhibin subunit cDNAs indicate that they are part of the TGF/3 gene family, which encodes a group of polypeptides with diverse regulatory effects on the growth and differentiation of numerous cell types (17). Activins rapidly increase intracellular levels of FSH in primary cultures of pituitary cells, suggesting that they affect the rate of FSH biosynthesis (13). Studies examining the effect of activin on steady
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ALEXANDER ET AL.
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state FSH/3 mRNA have shown that FSH/3 mRNA levels increase significantly as early as 4 h after activin administration, followed by increased FSH secretion (18, 19). Recently, quantitative studies in cultured rat pituitaries investigating FSH/3 mRNA degradation rates indicate that activin may exert its effect at the posttranscriptional level by increasing FSH/3 mRNA stability (20). The effects of activin on pituitary FSH biosynthesis have been characterized using primary rat pituitary cultures; however, nothing is known of the effects of activin on FSH biosynthesis and secretion in the human pituitary. The regulation of FSH biosynthesis and secretion in neoplastic cells also has not been explored. An understanding of the regulation of FSH/5 biosynthesis by glycoprotein hormone-producing pituitary adenomas may be important when considering potential therapies that control FSH production in these tumors. In this report we examined the effects of activin on gonadotropin biosynthesis and secretion in primary cultures of glycoprotein hormone-producing pituitary adenomas as a first step toward elucidating the mechanisms that control FSH secretion in these tumors.
Materials and Methods Clinical data Glycoprotein hormone-producing pituitary adenoma tissue was obtained from 12 patients undergoing transsphenoidal surgery who ranged in age from 37-74 yr, with a median age of 57 yr (see Table 1). All patients presented with visual field abnormalities, and a pituitary macroadenoma with extrasellar extension was confirmed by magnetic resonance imaging. Patients 1,8, and 12 also presented with amenorrhea. Preoperative serum levels of TSH and T4 were normal in all patients. Serum concentrations of GH and/or somatomedin-C were below normal range in all patients. PRL levels were slightly elevated in patients 1,3,8, and 12, consistent with pituitary stalk compression. Most patients had normal preoperative serum gonadotropin levels. Abnormally high serum levels of gonadotropins were detected in patients 10 (elevated FSH /?-subunit), 11 (elevated FSH and FSH /3-subunit), and 12 (elevated a-subunit). Immunocytochemistry was performed on pituitary tumor tissue using specific antibodies for LH0, FSHjS, TSH/3, a-subunit, PRL, GH, and ACTH (21). All tumors were positive for one or more gonadotropin subunits (Table 2). Immunocytochemistry for PRL, GH, and ACTH was negative in all tumors, while 5 of 12 tumors were positive for TSH/?. Pituitary tissue culture Normal pituitary glands were collected from 200 to 250-g mature Sprague-Dawley female rats (Charles River Laboratories, Cambridge, MA) and enzymatically dispersed as previously described (22). Cells were incubated for 72 h in 10% fetal calf serum/Dulbecco's Modified Eagle's Medium (DMEM) and allowed to attach to 60-mm culture dishes coated with bovine
JCE & M • 1991 Vol 72 • No 6
TABLE 1. Preoperative clinical data of patients with glycoprotein hormone-producing adenomas LH Patient Age a-Subunit Sex no. (yr) (/*g/L) (IU/L) 1 40 F 0.6 4.4 2 70 F 1.5 18.9 0.5 3 64 M 1.1 1.1 4 65 M 5.1 1.2 5 57 F 44.0 6 66 F 3.4 1.2 0.7 7 42 M 5.6 8 37 F 0.7 9.1 9 74 F 19.0 1.0 0.4 10 64 F 2.0 0.5 11 50 M 7.6 17.4 12 43 F 15.6 Normal serum levels Males Premenopausal females Postmenopausal females
FSH
FSH0
(IU/L)
(Mg/L)
PRL (/xg/L)
8.3
NA
103.0
40.5
2.3 0.3 0.6 3.1 1.4 0.3
4.0 6.5
89.2 9.2 3.9
14.2 28.8 11.0 56.6 19.7
0.8 1.9 16.6 1.1
0.5
4.5
17.0 12.8 25.0 53.9 14.0 76.0 10.9 40.0
10.0 109.0
0.5-2.5 0.5-2.5
3.0-18.0 0.81-19.2
Vol. 72, No. 6 Printed in U.S.A.
The Effects of Activin on Follicle-Stimulating Hormone Secretion and Biosynthesis in Human Glycoprotein Hormone-Producing Pituitary Adenomas* JOSEPH M. ALEXANDER, J. LARRY JAMESON, HELEN A. BIKKAL, RALPH H. SCHWALL, AND ANNE KLIBANSKI Neuroendocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114; and the Department of Developmental Biology, Genentech, Inc. (R.H.S.), South San Francisco, California 94080
ABSTRACT. The effects of activin on pituitary FSH biosynthesis have been previously characterized using primary rat pituitary cultures; however, little is known of the effects of activin on FSH biosynthesis and secretion in human pituitary tissue. Production of intact glycoprotein hormones and free subunits is increasingly recognized in pituitary tumors; however, the regulation of gonadotropins in such tumors has not been addressed. We have investigated the effects of human recombinant activin on glycoprotein hormone biosynthesis and secretion in primary cultures of 12 human glycoprotein hormone-producing pituitary adenomas and compared this with the effects of activin in normal rat anterior pituitary cells. In 33% of the human pituitary tumors studied, significant (P < 0.05) increases in FSH/3 secretion occurred in response to incubation with 20 ng/mL activin for 24 h (19-287% stimulation), without changes in the production of intact FSH. A Northern analysis performed on cells derived from one tumor indicated that FSH/3 mRNA
levels increased 350% after activin treatments; however, FSH secretion did not parallel the mRNA changes. None of the human glycoprotein hormone-producing tumors significantly increased FSH secretion in response to activin. To validate the biological activity of recombinant human activin-A and to confirm time and dose conditions for the human tumor cultures, we also examined its ability to stimulate FSH production in rat pituitary cultures. Activin (20 ng/mL) added to the culture medium significantly increased FSH secretion and steady state levels of FSHjS mRNA after 24 h. These data indicate that some glycoprotein hormone-producing pituitary tumors treated with purified activin have discordant responses of intact gonadotropins and free subunit responses. In contrast to responses in normal rat gonadotrophs, FSH/3 biosynthetic pathways may be uncoupled from intact FSH secretion in a subset of glycoprotein hormone-producing pituitary adenomas. (J Clin Endocrinol Metab 72: 1261-1267, 1991)
C
LINICALLY nonfunctioning pituitary tumors represent 25-30% of the diagnosed pituitary adenomas and often secrete intact glycoprotein hormones and/or their free subunits (1). Light and electron microscopy studies of these tumors show the presence of secretory granules in neoplastic pituitary tissue (2), and the majority of these tumors secrete FSH or free a- and FSH/3subunits (3-5). RNA analysis (6-9) and immunocytochemistry (10-12) also confirm the synthesis of intact gonadotropins or their subunits by a subset of clinically nonfunctioning pituitary adenomas. FSH production in these tumors has been well documented, but the biosynthetic mechanisms that regulate FSH production and secretion in neoplastic pituitary cells are poorly understood. There is increasing evidence that gonadal peptides play an important role in the regulation of normal pituiReceived September 27, 1990. Address requests for reprints to: Anne Klibanski, M.D., Neuroendocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. * This work was supported by NIDDK Grants DK-40947, DK-07028, and HD-23519.
tary FSH biosynthesis. Therefore, it is essential to characterize the responses of neoplastic pituitary tumors to peptides that have selective effects on FSH biosynthesis and secretion. Activins are polypeptides of gonadal origin which specifically stimulate FSH release by pituitary gonadotrophs (13). The activin polypeptides are dimers consisting of two inhibin /3-subunits (14, 15). The three forms of activin, activin-A (0A/3A), activin-B (/3B/3B), and activinAB (/3A/3B), have been shown to have similar biological activity. Activin-A and -AB have been purified from natural sources, while bioactive activin-A and B have been produced using recombinant DNA technology (16). Sequence analysis of cloned inhibin subunit cDNAs indicate that they are part of the TGF/3 gene family, which encodes a group of polypeptides with diverse regulatory effects on the growth and differentiation of numerous cell types (17). Activins rapidly increase intracellular levels of FSH in primary cultures of pituitary cells, suggesting that they affect the rate of FSH biosynthesis (13). Studies examining the effect of activin on steady
1261
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 23:54 For personal use only. No other uses without permission. . All rights reserved.
ALEXANDER ET AL.
1262
state FSH/3 mRNA have shown that FSH/3 mRNA levels increase significantly as early as 4 h after activin administration, followed by increased FSH secretion (18, 19). Recently, quantitative studies in cultured rat pituitaries investigating FSH/3 mRNA degradation rates indicate that activin may exert its effect at the posttranscriptional level by increasing FSH/3 mRNA stability (20). The effects of activin on pituitary FSH biosynthesis have been characterized using primary rat pituitary cultures; however, nothing is known of the effects of activin on FSH biosynthesis and secretion in the human pituitary. The regulation of FSH biosynthesis and secretion in neoplastic cells also has not been explored. An understanding of the regulation of FSH/5 biosynthesis by glycoprotein hormone-producing pituitary adenomas may be important when considering potential therapies that control FSH production in these tumors. In this report we examined the effects of activin on gonadotropin biosynthesis and secretion in primary cultures of glycoprotein hormone-producing pituitary adenomas as a first step toward elucidating the mechanisms that control FSH secretion in these tumors.
Materials and Methods Clinical data Glycoprotein hormone-producing pituitary adenoma tissue was obtained from 12 patients undergoing transsphenoidal surgery who ranged in age from 37-74 yr, with a median age of 57 yr (see Table 1). All patients presented with visual field abnormalities, and a pituitary macroadenoma with extrasellar extension was confirmed by magnetic resonance imaging. Patients 1,8, and 12 also presented with amenorrhea. Preoperative serum levels of TSH and T4 were normal in all patients. Serum concentrations of GH and/or somatomedin-C were below normal range in all patients. PRL levels were slightly elevated in patients 1,3,8, and 12, consistent with pituitary stalk compression. Most patients had normal preoperative serum gonadotropin levels. Abnormally high serum levels of gonadotropins were detected in patients 10 (elevated FSH /?-subunit), 11 (elevated FSH and FSH /3-subunit), and 12 (elevated a-subunit). Immunocytochemistry was performed on pituitary tumor tissue using specific antibodies for LH0, FSHjS, TSH/3, a-subunit, PRL, GH, and ACTH (21). All tumors were positive for one or more gonadotropin subunits (Table 2). Immunocytochemistry for PRL, GH, and ACTH was negative in all tumors, while 5 of 12 tumors were positive for TSH/?. Pituitary tissue culture Normal pituitary glands were collected from 200 to 250-g mature Sprague-Dawley female rats (Charles River Laboratories, Cambridge, MA) and enzymatically dispersed as previously described (22). Cells were incubated for 72 h in 10% fetal calf serum/Dulbecco's Modified Eagle's Medium (DMEM) and allowed to attach to 60-mm culture dishes coated with bovine
JCE & M • 1991 Vol 72 • No 6
TABLE 1. Preoperative clinical data of patients with glycoprotein hormone-producing adenomas LH Patient Age a-Subunit Sex no. (yr) (/*g/L) (IU/L) 1 40 F 0.6 4.4 2 70 F 1.5 18.9 0.5 3 64 M 1.1 1.1 4 65 M 5.1 1.2 5 57 F 44.0 6 66 F 3.4 1.2 0.7 7 42 M 5.6 8 37 F 0.7 9.1 9 74 F 19.0 1.0 0.4 10 64 F 2.0 0.5 11 50 M 7.6 17.4 12 43 F 15.6 Normal serum levels Males Premenopausal females Postmenopausal females
FSH
FSH0
(IU/L)
(Mg/L)
PRL (/xg/L)
8.3
NA
103.0
40.5
2.3 0.3 0.6 3.1 1.4 0.3
4.0 6.5
89.2 9.2 3.9
14.2 28.8 11.0 56.6 19.7
0.8 1.9 16.6 1.1
0.5
4.5
17.0 12.8 25.0 53.9 14.0 76.0 10.9 40.0
10.0 109.0
0.5-2.5 0.5-2.5
3.0-18.0 0.81-19.2
