0013-7227/91/1294-1721$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 4 Printed in U.S.A.
Activin Stabilizes Follicle-Stimulating Hormone-Beta Messenger Ribonucleic Acid Levels RONA S. CARROLL, ANNE Z. CORRIGAN, WYLIE VALE, AND WILLIAM W. CHIN Division of Genetics, Department of Medicine (R.S.C., W.W.C.), Brigham and Women's Hospital; Howard Hughes Medical Institute and Harvard Medical School; Boston, Massachusetts 02115; and Clayton Foundation Laboratories for Peptide Biology (A.Z.C., W. V.), The Salk Institute, La Jolla, California 92037
ABSTRACT. Activin, a gonadal peptide, stimulates FSH secretion in association with an increase in FSH/3 messenger RNA (mRNA) levels at the level of the anterior pituitary gland. The goal of these studies was to determine whether the effects of recombinant human activin A (rhActivin A) are exerted at the post-transcriptional level by affecting the stability of FSH/3 mRNA. We determined the apparent half-life of FSH/3 mRNA in the presence and absence of rhActivin A using actinomycin D. The anterior pituitary glands from adult male rats were isolated and dispersed enzymatically. Cells were preincubated in the presence of rhActivin A for 24 h to increase FSH/3 mRNA levels. Actinomycin D was then added and the cells were incubated for a subsequent 4, 6, 8, 12, and 24 h in the presence or absence of rhActivin A. As reported earlier, the addition of rhActivin A caused parallel increases in FSH secretion and
T
HE GONADAL peptides, inhibin and activin, are structurally related proteins originally isolated from follicular fluid and have been shown to regulate FSH secretion specifically (1-8). Inhibin is a heterodimer, composed of an a-chain, and one of two similar but distinct /3-chains (/3A or /3B)- Inhibin has been shown to decrease FSH secretion, FSH cell content, and FSH/? messenger RNA (mRNA) levels in primary pituitary cell cultures (9, 10). Two forms of activin have been isolated from follicular fluid (5-7) and shown to be either a disulfide-linked homodimer of the inhibin /3A (activin A) or a heterodimer composed of a /3A- and a /?B-subunit protein (activin AB). Activin B (a homodimer of/3B) has not yet been isolated from follicular fluid, but it has been prepared using recombinant DNA technology (11). All forms of activin have similar bioactivity, each stimulating FSH secretion from primary pituitary cell cultures. They inhibit secretion of growth hormone and growth of somatotropic cells (12), stimulate erythroid differentiation, enhance colony formation in bone marrow cultures (13, 14), and induce mesodermal differentiation (15). Received April 9, 1991. Address all correspondence and requests for reprints to: Dr. Rona Carroll, G. W. Thorn Research Building, Room 905, Brigham and Women's Hospital, 20 Shattuck Street, Boston, Massachusetts 02115.
FSH/3 mRNA levels, while having no effect on a or LH/3 mRNA levels. Actinomycin D treatment decreased FSH/3 mRNA to 49, 39, and 16% of control levels at the 4, 6, and 8 h time points, respectively. In contrast, when actinomycin D was added in the presence of rhActivin A FSH/3 mRNA was reduced to 80, 58, and 42% of control levels at the 4, 6, and 8 h time points, respectively. Using the least squares method of analysis, the apparent half-lives of FSH/3 mRNA under these two conditions were calculated. In the presence of actinomycin D, the half-life of FSH/3 mRNA was 3.1 h. The addition of activin significantly increased the half-life to 6.5 h. These results suggest that activin A stimulates FSH/3 mRNA levels, at least in part, at the posttranscriptional level by increasing the stability of FSH/3 mRNA. (Endocrinology 129: 1721-1726,1991)
Activin is also capable of inducing inhibin mRNA and protein expression as well as augmenting cAMP production in cultured granulosa cells (16). The cDNAs encoding the three known subunits (a, /3A, and /3B) have been cloned and sequenced (17, 18). The subunits have been detected in a number of tissues including the ovary, testes, placenta, brain, kidney, bone marrow, and pituitary (19, 20). Receptors for activin are expressed in a number of different cell types including the human leukemia cell line K562, mouse erythroleukemia cells, and follicular granulosa cells. Activin belongs to a family of growth and differentiation factors that includes transforming growth factor-/? (21), Mullerian inhibiting substance (22), the Xenopus protein Vgl (23), the product of the decapentaplegia gene complex in Drosophila (24), and several morphogenic proteins derived from bone (25). There is a high degree of conservation in the protein sequences among the members of this family suggesting that these proteins have important functions. We and others have previously shown that the action of recombinant human activin A (rhActivin A) stimulates FSH secretion and concomitantly increases FSH/3 mRNA levels with a direct effect on the anterior pituitary gland (9, 26). The goal of these studies was to determine 1721
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ACTIVIN STABILIZES FSH/3 mRNA LEVELS
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whether any part of the effect of rhActivin A is exerted at the posttranscriptional level by altering FSH/3 mRNA stability in primary anterior pituitary cell cultures.
tion to achieve a specific activity of 0.5-1 X 109 cpm//ug DNA (31).
Materials and Methods
Total RNA was extracted from dispersed rat pituitary cells and subunit levels mRNAs were determined using blot hybridization analysis. Cells per well (5 x 106) were harvested in 1 ml of 4 M guanidinium thiocyanate. The guanidinium thiocyanate was layered over 5.7 M CsCl and spun at 100,000 x g in a tabletop TL100 (Beckman Centrifuge, Fullerton, CA) for 16 h at 20 C. The RNA pellets were dissolved in 0.3 M sterile sodium acetate and the RNA was ethanol precipitated. Ten micrograms (A26o) of total RNA for each sample were subjected to electrophoresis and diffusion blotting onto nitrocellulose (32). Each blot was hybridized with labeled FSH/3 and LH/3, and then asubunit and /3-actin probes, using conditions previously described (29, 30). Blots were washed and subjected to autoradiography, and the band densities were determined by laser densitometry (Molecular Dynamics, Sunnyvale, CA).
Cell culture Anterior pituitary glands were dissected from mature male rats (Sprague-Dawley, 200-250 g; Bantin-Kingman, Fremont, CA) after decapitation (performed at the Salk Institute). After extensive rinsing in HEPES buffer [25 mM HEPES, 137 mM NaCl, 5 mM KC1, and 0.7 mM Na2HPO4 (pH 7.3)], hemisected pituitaries were dissociated as described previously (5, 27). Briefly, tissues were incubated with 0.4% (w/v) collagenase (type II, Worthington, Freehold, NJ) and deoxyribonuclease II (8000 U/ml; Sigma, St. Louis, MO) at 37 C for approximately 2 h in a 100 ml jacketed spinner flask, followed by incubation with 0.25% (w/v) Viokase (GIBCO, Grand Island, NY) at 37 C for 8 min. Dissociated cells were then washed and plated in /3PJ culture medium (Scientific Services, Salk Institute) with nystatin, Sato's cocktail [insulin (5 mg/1), transferrin (5 mg/1), parathyroid hormone (0.5 mg/1), T 3 (30 pM), and fibroblast growth factor (1 mg/1), and 2% (w/v) fetal bovine serum (HyClone)]. Cells were plated at a density of 5.0 x 106 cells in 60 mm tissue culture dishes (Falcon Plastics, Los Angeles, CA) and were allowed to attach for 3 days at 37 C in an incubator [5% CO2-95% air] before any treatment was initiated. On the third day, the cultures were washed with fresh medium supplemented with 2% fetal bovine serum, followed by the addition of rhActivin A (courtesy of Genentech) (28). Experimental procedures Recombinant human activin A was stored at 4 C in 0.05 M acetic acid. To verify that the rhActivin A was effective in increasing FSH/3 mRNA levels in these studies, cells were pretreated with either vehicle (0.05 M acetic acid) or rhActivin A for 24 h. To calculate the apparent half-life of the FSH/3 gene, all cells were pretreated with rhActivin A and subsequently treated with ethanol (0.5%, 25 ^1) or actinomycin D which was dissolved in ethanol and harvested 4, 6, 8, 12, and 24 h later. Gonadotropin RIAs FSH and LH values were measured by RIA with materials provided by the NIDDK using the FSH and LH RP-2 standards (29, 30). In the LH RIA, separation of the antigen-antibody complexes was achieved by the addition of Staphylococcal protein-A (Pansorbin, Calbiochem-Behring Corp., La Jolla, CA). DNA fragments The probes used for hybridization have been described previously (29, 30) and are: a 495 bp Ncol-Ncol rat a-subunit cDNA fragment, a 350 bp Pstl-Pstl rat LH/3 cDNA fragment, a 1000 bp Hindlll-EcoRl rat FSH/3 genomic DNA fragment, and a 650 bp Pstl-Pstl fragment of a mouse /3-actin cDNA clone (courtesy of Dr. Bruce Spiegelman),. The cDNA and genomic fragments were labeled using random primer transla-
Subunit mRNA determinations
Standardization of data The amount of RNA in each lane of each blot (10 ng by A26o) was internally standardized within a blot by assessing the amount of /3-actin mRNA per lane and correcting the a, LH/3, and FSH/3 mRNA levels accordingly, as described previously (29, 30). Two Northern blots were also internally standardized with cyclophilin mRNA (another housekeeping gene) in addition to /3-actin. The data normalized with cyclophilin correlated well with those obtained with /3-actin. Statistical analysis Linear regression analysis followed by t tests for independent samples was used to assess the statistical significance of changes in mRNA and gonadotropin levels. Z tests were used to assess the statistical change in the half-life of FSH/3 mRNA (33).
Results Effects of actinomycin D on activin-stimulated accumulation of FSHfi mRNA levels Male rats were killed and pituitary cells were dispersed as described in Materials and Methods. Cells were maintained in culture for 72 h after which rhActivin A (20 ng/ml) or medium alone was added to the cultures. Twenty-four hours later, actinomycin D (2 /xM) or ethanol was added to all the plates and the cells were incubated for a subsequent 4, 6, 8,12, or 24 h. RhActivin A pretreatment for 24 h was able to stimulate FSH/3 mRNA levels significantly, 2.5 to 3.0-fold (P < 0.05) over those seen in vehicle-treated controls (Fig. 1). Actinomycin D treatment for 4 h reduced the basal levels of FSH/3 mRNA to 49% of untreated controls. In contrast, when actinomycin D was added in the presence of rhActivin A, FSH/3 mRNA levels were 80% of control levels. At the 6- and 8-h time points FSH/3 mRNA levels
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ACTIVIN STABILIZES FSH/? mRNA LEVELS 10
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44 and 48% of controls, respectively. At these times LH/? mRNA levels were unchanged.
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Effects of actinomycin D on actiuin-stimulated FSH secretion
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FSH secretion was significantly increased 2.1-fold (P < 0.05) by 24 h of rhActivin A treatment over vehicletreated controls (Fig. 4). No effect on FSH secretion was seen with 4, 6, or 8 h of actinomycin D treatment. In contrast, at the 12- and 24-h time points, FSH secretion was 74 and 65% of controls in the presence of actinomycin D (P < 0.05), respectively. When actinomycin D was added in the presence of rhActivin A, FSH levels were stimulated 2.3- and 2.2-fold at the 12- and 24-h time points (P < 0.05) over those seen in the actinomycin D alone group. The addition of rhActivin A in the presence of actinomycin D also increased FSH levels above those in the vehicle-treated group by 1.7- and 1.5-fold (P < 0.05) at the 12- and 24-h time points, respectively (data not shown).
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Discussion Hours after Act D FIG. 1. Effects of rhActivin A in the presence of actinomycin D on FSH/3 subunit mRNA levels. Primary pituitary cell cultures were pretreated with rhActivin A (20 ng/ml) or medium alone 72 h after dispersion. Twenty-four hours later, actinomycin D (2 fiM) was added to all plates and the cells were incubated for a subsequent 4, 6, or 8 h. Cells were harvested and FSH/3 mRNA levels were determined. mRNA levels are expressed as arbitrary densitometric units (A.D.U.). All values represent mean ± SD, n = 2. ETOH, ethanol; Act D, actinomycin D. (*, P < 0.05, significantly different from ethanol-treated controls; +, P < 0.05, significantly different from act D group).
were 39% (P < 0.05) and 16% (P < 0.05) of controls, respectively, in the presence of actinomycin D. When actinomycin D was added in the presence of rhActivin A, FSH/? mRNA levels were 58 and 42% of controls, respectively. Using the least squares method of analysis and the 0-, 4-, 6-, and 8-h time points, the apparent halflives of FSH/3 mRNA in the presence and absence of rhActivin were calculated. In the presence of actinomycin D only, the half-life of FSH/? mRNA was 3.1 h. The addition of rhActivin A significantly increased the halflife to 6.5 h (P < 0.05) (Fig. 2). Twelve and twenty-four hours after actinomycin D treatment, FSH/? mRNA levels were undetectable in all groups by Northern blot analysis (data not shown). Basal a and LH/? mRNA levels were unaffected by treatment with actinomycin D, or rhActivin A and actinomycin D (Fig. 3) at the 4-, 6-, and 8-h time points. Twelve and twenty-four hours after actinomycin D treatment, a mRNA levels were decreased
We and others have shown that rhActivin A increases total intracellular FSH, FSH secretion, and steady state FSH/? mRNA levels in pituitary cell cultures (5, 9, 26). In our studies, the dose-response and time-course of the effects of rhActivin A on FSH secretion and steady state gonadotropin subunit mRNA levels were examined. RhActivin A at a dose of 1 ng/ml was able to stimulate FSH/3 mRNA levels and FSH secretion significantly. In a time-course experiment, a significant rise in FSH/? mRNA levels was noted as early as 4 h after rhActivin A administration and maintained for up to 24 h. However, a rise in FSH secretion was not observed until 8 h after rhActivin A addition. We did not observe any change in LH secretion or LH/? mRNA levels. By contrast, Attardi and Miklos (26) noted a small increase in secretion and cell content of LH, free glycoprotein asubunit, and a and LH/? mRNA levels. The mechanism by which activin stimulates FSH/? mRNA levels and FSH secretion is not known. We hypothesized that one focus of regulation is transcription. Accordingly, we performed nuclear run-on experiments using primary pituitary cells. With at least 10 attempts, we have been unable to obtain sufficient incorporation of radioactivity into nascent RNA in order to measure FSH/? gene transcription in this system. In contrast, we can readily detect both a and LH/? gene transcription. Thus, we cannot determine whether rhActivin A regulates FSH/? mRNA at the transcriptional level. We next hypothesized that rhActivin A stimulation of FSH/? mRNA may result, in part, by stabilization of the
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ACTIVIN STABILIZES FSH/? mRNA LEVELS
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100
FIG. 2. Apparent half-lives of rat FSH/3 mRNA in the presence and absence of actinomycin D. Primary pituitary cell cultures were pretreated 72 h after dispersion with rhActivin A (20 ng/ml) or medium alone. Twenty-four hours later, actinomycin D (2 pM) was added to all plates and the cells were incubated for a subsequent 4, 6, or 8 h. Cells were harvested and FSH/3 levels were determined. The values shown are relative to the ethanol (ETOH) mRNA levels at each time point. The ordinate is plotted on a logarithmic scale and the least squares fit of the data was used to determine the apparent FSH/3 mRNA half-lives. Act D, actinomycin D.
Endo• 1991 Voll29«No4
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E
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10 Hours after actinomycin D treatment A.
FIG. 3. Effects of rhActivin A in the presence of actinomycin D on a (A) and LHjS (B) subunit mRNA levels. Primary pituitary cell cultures were pretreated with rhActivin A (20 ng/ml) or medium alone 72 h after dispersion. Twenty-four hours later, actinomycin D (2 JIM) was added to all plates and the cells were incubated for a subsequent 4, 6, or 8 h. Cells were harvested, and a and LH0 mRNA levels were determined. mRNA levels are expressed as arbitrary densitometric units (A.D.U.). All values represent mean ± SD, n = 2. ETOH, ethanol; Act D, actinomycin D.
6-
Hours after Act D
mRNA. In this study we used actinomycin D to inhibit transcription and examined the decay rates of a, LH0, and FSH/? mRNA levels in the absence and presence of rhActivin A. There are drawbacks to the use of actinomycin D to determine mRNA half-lives because it is an inhibitor of RNA synthesis that may also affect cell metabolism. As a result, such a well described analysis provides only an apparent mRNA half-life. Other methods which can be used to examine mRNA half-lives include steady state labeling and the labeled pulse-chase approach. These methods would be very difficult to use in the cultured pituitary system since the level of pulse
label incorporation would be low. First, only 10% of the cells are gonadotropes and second, the rate of transcription of the FSH0 gene is low. Despite the limitations of the actinomycin D method, we have shown that activin stimulates FSH/3 mRNA levels and FSH secretion posttranscriptionally by increasing the stability of the FSH/3 mRNA without affecting a and LH/3 mRNA levels. The first experiments we performed examined the apparent half-life of the FSH0 gene in vitro in dispersed male rat anterior pituitary cell cultures. In this experimental paradigm, rhActivin A was still able to significantly stimulate FSH/J mRNA levels after inhibition of
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ACTIVIN STABILIZES FSH0 mRNA LEVELS 200
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FlG. 4. Effects of rhActivin A in the presence of actinomycin D on FSH secretion. Primary pituitary cell cultures were pretreated with rhActivin A (20 ng/ml) or medium alone 72 h after dispersion. Twentyfour hours later, actinomycin D (2 ^M) was added to all plates and the cells were incubated for a subsequent 4, 6, or 8 h. Media levels of FSH were determined by RIA. All values represent mean ± SD, n = 2. ETOH, ethanol; Act D, actinomycin D. (*, P < 0.05, significantly different from ethanol-treated controls)
RNA synthesis by actinomycin D (2 ^M) at the 4-, 6-, and 8-h time points. The apparent half-life of FSH/? was 3.1 h. The addition of rhActivin A significantly increased the apparent half-life to 6.5 h. RhActivin A was also able to stimulate FSH levels significantly in the presence of actinomycin D at the 12-h and 24-h time points. In contrast, studies by Schwall et al. (28) showed that actinomycin D (1 /*M) completely blocked the stimulatory response to rhActivin A, but not to gonadotropin-releasing hormone in cultured pituitary cells. The apparent discrepancy of the effects of activin in the presence of actinomycin D in these two studies could be due to the differences in the age of the animals [adult males (the present study) vs. 21-day-old females] or the length of time of the actinomycin D treatment [24 h (the present study) vs. 48 h]. It is not likely that our observations reflect an inadequate dose of actinomycin D in this study because, in fact, we used a higher dose [2 /iM (the present study) vs. 1 fiM (previous study)]. The mechanisms for mRNA stabilization by activin are not known. Recently, much attention has been fo-
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cused on the factors that influence mRNA stability. Shaw and Kamen (34) described a specific sequence AUUUA that confers mRNA instability to many shortlived mRNAs such as those encoding GM-CSF. The introduction of this sequence into stable mRNAs such as /?-globin causes the RNA to become highly unstable. This AU-rich sequence has been demonstrated to be partially responsible for instability of the RNA by binding a specific protein (35). However, it should be noted that the 3'-untranslated region of the rat FSH/? mRNA has no such AUUUA sequence. Another example of such elements can be found in the 3'-untranslated region of the transferrin receptor mRNA that includes a specific sequence that plays a role in iron-dependent degradation ofthemRNA(36). There are also examples of hormone-induced mRNA stabilization. The mRNA encoding vitellogenin is stabilized 30-fold by estrogen treatment (37). Similarly, in cultured mammary epithelial cells, the combination of insulin, hydrocortisone, and prolactin treatment stimulates transcription of the gene encoding the milk protein casein by only 3-fold, while post-transcriptional events are responsible for a 25-fold increase in mRNA levels (38). Krane et al. (39) showed that the levels of TSH/? mRNA are also regulated posttranscriptionally by T3induced changes in subunit mRNA half-life. The change in half-life appears to be associated with a reduction in the size of the poly(A) tail. Last, a recent study (40) in which the disappearance of gonadotropin mRNAs was examined in vivo, suggests that testosterone may increase FSH/? mRNA levels by prolonging the half-life of the FSH/? mRNA. In summary, we have shown that rhActivin A increases steady state levels of FSH/? mRNA, at least in part, by increasing the stability of the FSH/? mRNA directly at the level of the anterior pituitary gland. It is not yet clear whether activin may also increase FSH secretion by increasing transcription of the FSH/? gene.
References 1. Ling N, Ying S-Y, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:7217-7221 2. Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun 133:120-127 3. Miyamoto K, Hasegawa Y, Fukuda M, Nomura M, Igarashi M, Kangawa K, Matsuo H 1985 Isolation of porcine follicular fluid inhibin of 32K daltons. Biochem Biophys Res Commun 133:120127 4. Robertson DM, Foulds LM, Leversha L, Morgan FJ, Hearn MTW, Burger HG, Wettenhall REH, de Kretser DM 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun 126:220-226 5. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature
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321:776-779 6. Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the betasubunits from the two forms of inhibin. Nature 321:779-782 7. Ling N, Ying S-Y, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R 1986 A homodimer of the subunits of inhibin A stimulates the secretion of pituitary follicle-stimulating hormone. Biochem Biophys Res Commun 138:1129-1137 8. Vale W, Hsueh A, Rivier C, Yu J, 1990 The inhibin/activin family of hormones and growth factor. In: Born GVR, Cuatrecasas P, Herken H (eds) Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors II, Springer-Verlag, Berlin, pp 211-248 9. Carroll RS, Corrigan AZ, Gharib SD, Vale W, Chin WW 1989 Inhibin, activin and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels. Mol Endocrinol 3:19691976 10. Attardi B, Keeping HS, Winter SJ, Kotsuji F, Maurer RA, Troen P 1989 Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone /3 by inhibin from primate Sertoli cells. Mol Endocrinol 3:280-287 11. Mason AJ, Berkemeier LM, Schmelzer CH, Schwall RH 1989 Activin B: precursor sequences, genomic structure and in vitro activities. Mol Endocrinol 3:1352-1358 12. Billestrup N, Gonzalez-Manchon C, Potter E, Vale W 1990 Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro. Mol Endocrinol 4:356-362 13. Eto Y, Tsuji T, Takezawa M, Takano S, Yokogawa Y, Shibai H 1987 Purification and characterization of erythroid differentiation factor (EDF) isolated from human leukemia cell line THP-1. Biochem Biophys Res Commun 142:1095-1103 14. Yu J, Shao L, Lemas V, Yu AL, Vaughan J, Rivier J, Vale W 1987 Importance of FSH-releasing protein and inhibin in erythrodifferentiation. Nature 330:765-767 15. Thomsen G, Woolf T, Whitman M, Sokol S, Vaughan J, Vale W, Melton DA 1990 Activins are expressed early in Xenopus embroygenesis and can induce axial mesoderm and anterior structures. Cell 63:485-493 16. LaPolt PS, Soto D, Su J-G, Campen CA, Vaughan J, Vale W, Hsueh A 1989 Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells. Mol Endocrinol 3:1666-1673 17. Woodruff T, Meunier H, Jones P, Hsueh A, Mayo K 1987 Rat inhibin: molecular cloning of a- and /3-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 1:561-568 18. Esch FS, Shimasaki S, Cooksey K, Mercado M, Mason AJ, Ying SY, Ueno N, Ling N 1987 Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinology 1:388-396 19. Meunier H, Rivier C, Evans R, Vale W 1988 Gonadal and extragonadal expression of inhibin a, /3A and j8B subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247251 20. Roberts V, Meunier H, Vaughan J, Rivier J, Rivier C, Vale W, Sawchenko P 1989 Production and regulation of inhibin subunits in pituitary gonadotropes. Endocrinology 124:552-554 21. Sporn MB, Roberts AB, Wakefield LM, Assoian RK 1986 Transforming growth factor-/?: biological function and chemical structure. Science 233:532-534
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22. Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung A, Ninfa EG, Frey AT, Gash DJ, Chow EP, Fisher RA, Bertonis JM, Torres G, Wallner BP, Ramachandran KL, Ragin RC, Manganaro TF, MacLaughlin DT, Donahoe PK 1986 Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human genes in animal cells. Cell 45:685-698 23. Weeks D, Melton D 1987 A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF/3. Cell 51:861-867 24. Padgett R, St Johnson R, Gelbart W 1987 A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-/? family. Nature 325:81-84 25. Wozney J, Rosen V, Celeste A, Mitsock L, Whitters M, Kriz R, Hewick R, Wang E 1988 Novel regulators of bone formation: molecular clones and activities. Science 242:1528-1534 26. Attardi B, Miklos J 1990 Rapid stimulatory effect of activin-A on messenger RNA encoding the follicle-stimulating hormone /3-subunit in rat pituitary cell cultures. Mol Endocrinol 4:721-726 27. Vale W, Vaughan J, Yamamoto G, Bruhn T, Douglas C, Dalton D, Rivier C, Rivier J 1983 Assay of corticotropin releasing factor. Methods Enzymol 103:565-577 28. Schwall RH, Nikolics K, Szony E, Gorman C, Mason AJ 1988 Recombinant expression and characterization of human activin A. Mol Endocrinol 2:1237-1242 29. Gharib SD, Bowers SM, Need LR, Chin WW 1986 Regulation of rat luteinizing hormone subunit messenger ribonucleic acids by gonadal steroid hormones. J Clin Invest 77:582-589 30. Gharib SD, Wierman ME, Badger TM, Chin WW 1987 Sex steroid hormone regulation of follicle-stimulating hormone subunit messenger ribonucleic acid (mRNA) levels in the rat. J Clin Invest 80:294-299 31. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction fragments to high specific activity. Anal Biochem 132:6— 13 32. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5205 33. Bruning JL, Kintz BL 1977 Computational Handbook of Statistics. Scott, Foresman and Co, Dallas 34. Shaw G, Kamen R 1986 A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659-667 35. Malter JS 1989 Identification of an AUUUA-specific messenger RNA binding protein. Science 246:664-666 36. Casey JL, Hentz MW, Koeller DM, Caughman SW, Rouault TA, Klausner RD, Harford JB 1988. Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation. Science 240:924-928 37. Brock ML, Shapiro DJ 1983 Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell 34:207-214 38. Eisenstein RS, Rosen JM 1988 Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level. Mol Cell Biol 8:3183-3190 39. Krane I, Spindel ER, Chin WW 1991 Thyroid hormone decreases the stability and the poly A tract length of rat thyrotropin /3 subunit mRNA. Mol Endocrinol 5:469-475 40. Paul SJ, Ortolano GA, Haisenleder DJ, Stewart JM, Shupnik MA, Marshall JC 1990 Gonadotropin subunit disappearance after blockade of GnRH action: testosterone prolongs the availability of FSH/3 subunit mRNA. Mol Endocrinol 4:1943-1955
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Vol. 129, No. 4 Printed in U.S.A.
Activin Stabilizes Follicle-Stimulating Hormone-Beta Messenger Ribonucleic Acid Levels RONA S. CARROLL, ANNE Z. CORRIGAN, WYLIE VALE, AND WILLIAM W. CHIN Division of Genetics, Department of Medicine (R.S.C., W.W.C.), Brigham and Women's Hospital; Howard Hughes Medical Institute and Harvard Medical School; Boston, Massachusetts 02115; and Clayton Foundation Laboratories for Peptide Biology (A.Z.C., W. V.), The Salk Institute, La Jolla, California 92037
ABSTRACT. Activin, a gonadal peptide, stimulates FSH secretion in association with an increase in FSH/3 messenger RNA (mRNA) levels at the level of the anterior pituitary gland. The goal of these studies was to determine whether the effects of recombinant human activin A (rhActivin A) are exerted at the post-transcriptional level by affecting the stability of FSH/3 mRNA. We determined the apparent half-life of FSH/3 mRNA in the presence and absence of rhActivin A using actinomycin D. The anterior pituitary glands from adult male rats were isolated and dispersed enzymatically. Cells were preincubated in the presence of rhActivin A for 24 h to increase FSH/3 mRNA levels. Actinomycin D was then added and the cells were incubated for a subsequent 4, 6, 8, 12, and 24 h in the presence or absence of rhActivin A. As reported earlier, the addition of rhActivin A caused parallel increases in FSH secretion and
T
HE GONADAL peptides, inhibin and activin, are structurally related proteins originally isolated from follicular fluid and have been shown to regulate FSH secretion specifically (1-8). Inhibin is a heterodimer, composed of an a-chain, and one of two similar but distinct /3-chains (/3A or /3B)- Inhibin has been shown to decrease FSH secretion, FSH cell content, and FSH/? messenger RNA (mRNA) levels in primary pituitary cell cultures (9, 10). Two forms of activin have been isolated from follicular fluid (5-7) and shown to be either a disulfide-linked homodimer of the inhibin /3A (activin A) or a heterodimer composed of a /3A- and a /?B-subunit protein (activin AB). Activin B (a homodimer of/3B) has not yet been isolated from follicular fluid, but it has been prepared using recombinant DNA technology (11). All forms of activin have similar bioactivity, each stimulating FSH secretion from primary pituitary cell cultures. They inhibit secretion of growth hormone and growth of somatotropic cells (12), stimulate erythroid differentiation, enhance colony formation in bone marrow cultures (13, 14), and induce mesodermal differentiation (15). Received April 9, 1991. Address all correspondence and requests for reprints to: Dr. Rona Carroll, G. W. Thorn Research Building, Room 905, Brigham and Women's Hospital, 20 Shattuck Street, Boston, Massachusetts 02115.
FSH/3 mRNA levels, while having no effect on a or LH/3 mRNA levels. Actinomycin D treatment decreased FSH/3 mRNA to 49, 39, and 16% of control levels at the 4, 6, and 8 h time points, respectively. In contrast, when actinomycin D was added in the presence of rhActivin A FSH/3 mRNA was reduced to 80, 58, and 42% of control levels at the 4, 6, and 8 h time points, respectively. Using the least squares method of analysis, the apparent half-lives of FSH/3 mRNA under these two conditions were calculated. In the presence of actinomycin D, the half-life of FSH/3 mRNA was 3.1 h. The addition of activin significantly increased the half-life to 6.5 h. These results suggest that activin A stimulates FSH/3 mRNA levels, at least in part, at the posttranscriptional level by increasing the stability of FSH/3 mRNA. (Endocrinology 129: 1721-1726,1991)
Activin is also capable of inducing inhibin mRNA and protein expression as well as augmenting cAMP production in cultured granulosa cells (16). The cDNAs encoding the three known subunits (a, /3A, and /3B) have been cloned and sequenced (17, 18). The subunits have been detected in a number of tissues including the ovary, testes, placenta, brain, kidney, bone marrow, and pituitary (19, 20). Receptors for activin are expressed in a number of different cell types including the human leukemia cell line K562, mouse erythroleukemia cells, and follicular granulosa cells. Activin belongs to a family of growth and differentiation factors that includes transforming growth factor-/? (21), Mullerian inhibiting substance (22), the Xenopus protein Vgl (23), the product of the decapentaplegia gene complex in Drosophila (24), and several morphogenic proteins derived from bone (25). There is a high degree of conservation in the protein sequences among the members of this family suggesting that these proteins have important functions. We and others have previously shown that the action of recombinant human activin A (rhActivin A) stimulates FSH secretion and concomitantly increases FSH/3 mRNA levels with a direct effect on the anterior pituitary gland (9, 26). The goal of these studies was to determine 1721
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ACTIVIN STABILIZES FSH/3 mRNA LEVELS
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whether any part of the effect of rhActivin A is exerted at the posttranscriptional level by altering FSH/3 mRNA stability in primary anterior pituitary cell cultures.
tion to achieve a specific activity of 0.5-1 X 109 cpm//ug DNA (31).
Materials and Methods
Total RNA was extracted from dispersed rat pituitary cells and subunit levels mRNAs were determined using blot hybridization analysis. Cells per well (5 x 106) were harvested in 1 ml of 4 M guanidinium thiocyanate. The guanidinium thiocyanate was layered over 5.7 M CsCl and spun at 100,000 x g in a tabletop TL100 (Beckman Centrifuge, Fullerton, CA) for 16 h at 20 C. The RNA pellets were dissolved in 0.3 M sterile sodium acetate and the RNA was ethanol precipitated. Ten micrograms (A26o) of total RNA for each sample were subjected to electrophoresis and diffusion blotting onto nitrocellulose (32). Each blot was hybridized with labeled FSH/3 and LH/3, and then asubunit and /3-actin probes, using conditions previously described (29, 30). Blots were washed and subjected to autoradiography, and the band densities were determined by laser densitometry (Molecular Dynamics, Sunnyvale, CA).
Cell culture Anterior pituitary glands were dissected from mature male rats (Sprague-Dawley, 200-250 g; Bantin-Kingman, Fremont, CA) after decapitation (performed at the Salk Institute). After extensive rinsing in HEPES buffer [25 mM HEPES, 137 mM NaCl, 5 mM KC1, and 0.7 mM Na2HPO4 (pH 7.3)], hemisected pituitaries were dissociated as described previously (5, 27). Briefly, tissues were incubated with 0.4% (w/v) collagenase (type II, Worthington, Freehold, NJ) and deoxyribonuclease II (8000 U/ml; Sigma, St. Louis, MO) at 37 C for approximately 2 h in a 100 ml jacketed spinner flask, followed by incubation with 0.25% (w/v) Viokase (GIBCO, Grand Island, NY) at 37 C for 8 min. Dissociated cells were then washed and plated in /3PJ culture medium (Scientific Services, Salk Institute) with nystatin, Sato's cocktail [insulin (5 mg/1), transferrin (5 mg/1), parathyroid hormone (0.5 mg/1), T 3 (30 pM), and fibroblast growth factor (1 mg/1), and 2% (w/v) fetal bovine serum (HyClone)]. Cells were plated at a density of 5.0 x 106 cells in 60 mm tissue culture dishes (Falcon Plastics, Los Angeles, CA) and were allowed to attach for 3 days at 37 C in an incubator [5% CO2-95% air] before any treatment was initiated. On the third day, the cultures were washed with fresh medium supplemented with 2% fetal bovine serum, followed by the addition of rhActivin A (courtesy of Genentech) (28). Experimental procedures Recombinant human activin A was stored at 4 C in 0.05 M acetic acid. To verify that the rhActivin A was effective in increasing FSH/3 mRNA levels in these studies, cells were pretreated with either vehicle (0.05 M acetic acid) or rhActivin A for 24 h. To calculate the apparent half-life of the FSH/3 gene, all cells were pretreated with rhActivin A and subsequently treated with ethanol (0.5%, 25 ^1) or actinomycin D which was dissolved in ethanol and harvested 4, 6, 8, 12, and 24 h later. Gonadotropin RIAs FSH and LH values were measured by RIA with materials provided by the NIDDK using the FSH and LH RP-2 standards (29, 30). In the LH RIA, separation of the antigen-antibody complexes was achieved by the addition of Staphylococcal protein-A (Pansorbin, Calbiochem-Behring Corp., La Jolla, CA). DNA fragments The probes used for hybridization have been described previously (29, 30) and are: a 495 bp Ncol-Ncol rat a-subunit cDNA fragment, a 350 bp Pstl-Pstl rat LH/3 cDNA fragment, a 1000 bp Hindlll-EcoRl rat FSH/3 genomic DNA fragment, and a 650 bp Pstl-Pstl fragment of a mouse /3-actin cDNA clone (courtesy of Dr. Bruce Spiegelman),. The cDNA and genomic fragments were labeled using random primer transla-
Subunit mRNA determinations
Standardization of data The amount of RNA in each lane of each blot (10 ng by A26o) was internally standardized within a blot by assessing the amount of /3-actin mRNA per lane and correcting the a, LH/3, and FSH/3 mRNA levels accordingly, as described previously (29, 30). Two Northern blots were also internally standardized with cyclophilin mRNA (another housekeeping gene) in addition to /3-actin. The data normalized with cyclophilin correlated well with those obtained with /3-actin. Statistical analysis Linear regression analysis followed by t tests for independent samples was used to assess the statistical significance of changes in mRNA and gonadotropin levels. Z tests were used to assess the statistical change in the half-life of FSH/3 mRNA (33).
Results Effects of actinomycin D on activin-stimulated accumulation of FSHfi mRNA levels Male rats were killed and pituitary cells were dispersed as described in Materials and Methods. Cells were maintained in culture for 72 h after which rhActivin A (20 ng/ml) or medium alone was added to the cultures. Twenty-four hours later, actinomycin D (2 /xM) or ethanol was added to all the plates and the cells were incubated for a subsequent 4, 6, 8,12, or 24 h. RhActivin A pretreatment for 24 h was able to stimulate FSH/3 mRNA levels significantly, 2.5 to 3.0-fold (P < 0.05) over those seen in vehicle-treated controls (Fig. 1). Actinomycin D treatment for 4 h reduced the basal levels of FSH/3 mRNA to 49% of untreated controls. In contrast, when actinomycin D was added in the presence of rhActivin A, FSH/3 mRNA levels were 80% of control levels. At the 6- and 8-h time points FSH/3 mRNA levels
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ACTIVIN STABILIZES FSH/? mRNA LEVELS 10
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44 and 48% of controls, respectively. At these times LH/? mRNA levels were unchanged.
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Effects of actinomycin D on actiuin-stimulated FSH secretion
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FSH secretion was significantly increased 2.1-fold (P < 0.05) by 24 h of rhActivin A treatment over vehicletreated controls (Fig. 4). No effect on FSH secretion was seen with 4, 6, or 8 h of actinomycin D treatment. In contrast, at the 12- and 24-h time points, FSH secretion was 74 and 65% of controls in the presence of actinomycin D (P < 0.05), respectively. When actinomycin D was added in the presence of rhActivin A, FSH levels were stimulated 2.3- and 2.2-fold at the 12- and 24-h time points (P < 0.05) over those seen in the actinomycin D alone group. The addition of rhActivin A in the presence of actinomycin D also increased FSH levels above those in the vehicle-treated group by 1.7- and 1.5-fold (P < 0.05) at the 12- and 24-h time points, respectively (data not shown).
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Discussion Hours after Act D FIG. 1. Effects of rhActivin A in the presence of actinomycin D on FSH/3 subunit mRNA levels. Primary pituitary cell cultures were pretreated with rhActivin A (20 ng/ml) or medium alone 72 h after dispersion. Twenty-four hours later, actinomycin D (2 fiM) was added to all plates and the cells were incubated for a subsequent 4, 6, or 8 h. Cells were harvested and FSH/3 mRNA levels were determined. mRNA levels are expressed as arbitrary densitometric units (A.D.U.). All values represent mean ± SD, n = 2. ETOH, ethanol; Act D, actinomycin D. (*, P < 0.05, significantly different from ethanol-treated controls; +, P < 0.05, significantly different from act D group).
were 39% (P < 0.05) and 16% (P < 0.05) of controls, respectively, in the presence of actinomycin D. When actinomycin D was added in the presence of rhActivin A, FSH/? mRNA levels were 58 and 42% of controls, respectively. Using the least squares method of analysis and the 0-, 4-, 6-, and 8-h time points, the apparent halflives of FSH/3 mRNA in the presence and absence of rhActivin were calculated. In the presence of actinomycin D only, the half-life of FSH/? mRNA was 3.1 h. The addition of rhActivin A significantly increased the halflife to 6.5 h (P < 0.05) (Fig. 2). Twelve and twenty-four hours after actinomycin D treatment, FSH/? mRNA levels were undetectable in all groups by Northern blot analysis (data not shown). Basal a and LH/? mRNA levels were unaffected by treatment with actinomycin D, or rhActivin A and actinomycin D (Fig. 3) at the 4-, 6-, and 8-h time points. Twelve and twenty-four hours after actinomycin D treatment, a mRNA levels were decreased
We and others have shown that rhActivin A increases total intracellular FSH, FSH secretion, and steady state FSH/? mRNA levels in pituitary cell cultures (5, 9, 26). In our studies, the dose-response and time-course of the effects of rhActivin A on FSH secretion and steady state gonadotropin subunit mRNA levels were examined. RhActivin A at a dose of 1 ng/ml was able to stimulate FSH/3 mRNA levels and FSH secretion significantly. In a time-course experiment, a significant rise in FSH/? mRNA levels was noted as early as 4 h after rhActivin A administration and maintained for up to 24 h. However, a rise in FSH secretion was not observed until 8 h after rhActivin A addition. We did not observe any change in LH secretion or LH/? mRNA levels. By contrast, Attardi and Miklos (26) noted a small increase in secretion and cell content of LH, free glycoprotein asubunit, and a and LH/? mRNA levels. The mechanism by which activin stimulates FSH/? mRNA levels and FSH secretion is not known. We hypothesized that one focus of regulation is transcription. Accordingly, we performed nuclear run-on experiments using primary pituitary cells. With at least 10 attempts, we have been unable to obtain sufficient incorporation of radioactivity into nascent RNA in order to measure FSH/? gene transcription in this system. In contrast, we can readily detect both a and LH/? gene transcription. Thus, we cannot determine whether rhActivin A regulates FSH/? mRNA at the transcriptional level. We next hypothesized that rhActivin A stimulation of FSH/? mRNA may result, in part, by stabilization of the
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ACTIVIN STABILIZES FSH/? mRNA LEVELS
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100
FIG. 2. Apparent half-lives of rat FSH/3 mRNA in the presence and absence of actinomycin D. Primary pituitary cell cultures were pretreated 72 h after dispersion with rhActivin A (20 ng/ml) or medium alone. Twenty-four hours later, actinomycin D (2 pM) was added to all plates and the cells were incubated for a subsequent 4, 6, or 8 h. Cells were harvested and FSH/3 levels were determined. The values shown are relative to the ethanol (ETOH) mRNA levels at each time point. The ordinate is plotted on a logarithmic scale and the least squares fit of the data was used to determine the apparent FSH/3 mRNA half-lives. Act D, actinomycin D.
Endo• 1991 Voll29«No4
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D A
E
ACTD ACTD+ACTIVIN
t1/2=3.1h
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10
10 Hours after actinomycin D treatment A.
FIG. 3. Effects of rhActivin A in the presence of actinomycin D on a (A) and LHjS (B) subunit mRNA levels. Primary pituitary cell cultures were pretreated with rhActivin A (20 ng/ml) or medium alone 72 h after dispersion. Twenty-four hours later, actinomycin D (2 JIM) was added to all plates and the cells were incubated for a subsequent 4, 6, or 8 h. Cells were harvested, and a and LH0 mRNA levels were determined. mRNA levels are expressed as arbitrary densitometric units (A.D.U.). All values represent mean ± SD, n = 2. ETOH, ethanol; Act D, actinomycin D.
6-
Hours after Act D
mRNA. In this study we used actinomycin D to inhibit transcription and examined the decay rates of a, LH0, and FSH/? mRNA levels in the absence and presence of rhActivin A. There are drawbacks to the use of actinomycin D to determine mRNA half-lives because it is an inhibitor of RNA synthesis that may also affect cell metabolism. As a result, such a well described analysis provides only an apparent mRNA half-life. Other methods which can be used to examine mRNA half-lives include steady state labeling and the labeled pulse-chase approach. These methods would be very difficult to use in the cultured pituitary system since the level of pulse
label incorporation would be low. First, only 10% of the cells are gonadotropes and second, the rate of transcription of the FSH0 gene is low. Despite the limitations of the actinomycin D method, we have shown that activin stimulates FSH/3 mRNA levels and FSH secretion posttranscriptionally by increasing the stability of the FSH/3 mRNA without affecting a and LH/3 mRNA levels. The first experiments we performed examined the apparent half-life of the FSH0 gene in vitro in dispersed male rat anterior pituitary cell cultures. In this experimental paradigm, rhActivin A was still able to significantly stimulate FSH/J mRNA levels after inhibition of
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ACTIVIN STABILIZES FSH0 mRNA LEVELS 200
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FlG. 4. Effects of rhActivin A in the presence of actinomycin D on FSH secretion. Primary pituitary cell cultures were pretreated with rhActivin A (20 ng/ml) or medium alone 72 h after dispersion. Twentyfour hours later, actinomycin D (2 ^M) was added to all plates and the cells were incubated for a subsequent 4, 6, or 8 h. Media levels of FSH were determined by RIA. All values represent mean ± SD, n = 2. ETOH, ethanol; Act D, actinomycin D. (*, P < 0.05, significantly different from ethanol-treated controls)
RNA synthesis by actinomycin D (2 ^M) at the 4-, 6-, and 8-h time points. The apparent half-life of FSH/? was 3.1 h. The addition of rhActivin A significantly increased the apparent half-life to 6.5 h. RhActivin A was also able to stimulate FSH levels significantly in the presence of actinomycin D at the 12-h and 24-h time points. In contrast, studies by Schwall et al. (28) showed that actinomycin D (1 /*M) completely blocked the stimulatory response to rhActivin A, but not to gonadotropin-releasing hormone in cultured pituitary cells. The apparent discrepancy of the effects of activin in the presence of actinomycin D in these two studies could be due to the differences in the age of the animals [adult males (the present study) vs. 21-day-old females] or the length of time of the actinomycin D treatment [24 h (the present study) vs. 48 h]. It is not likely that our observations reflect an inadequate dose of actinomycin D in this study because, in fact, we used a higher dose [2 /iM (the present study) vs. 1 fiM (previous study)]. The mechanisms for mRNA stabilization by activin are not known. Recently, much attention has been fo-
1725
cused on the factors that influence mRNA stability. Shaw and Kamen (34) described a specific sequence AUUUA that confers mRNA instability to many shortlived mRNAs such as those encoding GM-CSF. The introduction of this sequence into stable mRNAs such as /?-globin causes the RNA to become highly unstable. This AU-rich sequence has been demonstrated to be partially responsible for instability of the RNA by binding a specific protein (35). However, it should be noted that the 3'-untranslated region of the rat FSH/? mRNA has no such AUUUA sequence. Another example of such elements can be found in the 3'-untranslated region of the transferrin receptor mRNA that includes a specific sequence that plays a role in iron-dependent degradation ofthemRNA(36). There are also examples of hormone-induced mRNA stabilization. The mRNA encoding vitellogenin is stabilized 30-fold by estrogen treatment (37). Similarly, in cultured mammary epithelial cells, the combination of insulin, hydrocortisone, and prolactin treatment stimulates transcription of the gene encoding the milk protein casein by only 3-fold, while post-transcriptional events are responsible for a 25-fold increase in mRNA levels (38). Krane et al. (39) showed that the levels of TSH/? mRNA are also regulated posttranscriptionally by T3induced changes in subunit mRNA half-life. The change in half-life appears to be associated with a reduction in the size of the poly(A) tail. Last, a recent study (40) in which the disappearance of gonadotropin mRNAs was examined in vivo, suggests that testosterone may increase FSH/? mRNA levels by prolonging the half-life of the FSH/? mRNA. In summary, we have shown that rhActivin A increases steady state levels of FSH/? mRNA, at least in part, by increasing the stability of the FSH/? mRNA directly at the level of the anterior pituitary gland. It is not yet clear whether activin may also increase FSH secretion by increasing transcription of the FSH/? gene.
References 1. Ling N, Ying S-Y, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:7217-7221 2. Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun 133:120-127 3. Miyamoto K, Hasegawa Y, Fukuda M, Nomura M, Igarashi M, Kangawa K, Matsuo H 1985 Isolation of porcine follicular fluid inhibin of 32K daltons. Biochem Biophys Res Commun 133:120127 4. Robertson DM, Foulds LM, Leversha L, Morgan FJ, Hearn MTW, Burger HG, Wettenhall REH, de Kretser DM 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun 126:220-226 5. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature
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321:776-779 6. Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the betasubunits from the two forms of inhibin. Nature 321:779-782 7. Ling N, Ying S-Y, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R 1986 A homodimer of the subunits of inhibin A stimulates the secretion of pituitary follicle-stimulating hormone. Biochem Biophys Res Commun 138:1129-1137 8. Vale W, Hsueh A, Rivier C, Yu J, 1990 The inhibin/activin family of hormones and growth factor. In: Born GVR, Cuatrecasas P, Herken H (eds) Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors II, Springer-Verlag, Berlin, pp 211-248 9. Carroll RS, Corrigan AZ, Gharib SD, Vale W, Chin WW 1989 Inhibin, activin and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels. Mol Endocrinol 3:19691976 10. Attardi B, Keeping HS, Winter SJ, Kotsuji F, Maurer RA, Troen P 1989 Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone /3 by inhibin from primate Sertoli cells. Mol Endocrinol 3:280-287 11. Mason AJ, Berkemeier LM, Schmelzer CH, Schwall RH 1989 Activin B: precursor sequences, genomic structure and in vitro activities. Mol Endocrinol 3:1352-1358 12. Billestrup N, Gonzalez-Manchon C, Potter E, Vale W 1990 Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro. Mol Endocrinol 4:356-362 13. Eto Y, Tsuji T, Takezawa M, Takano S, Yokogawa Y, Shibai H 1987 Purification and characterization of erythroid differentiation factor (EDF) isolated from human leukemia cell line THP-1. Biochem Biophys Res Commun 142:1095-1103 14. Yu J, Shao L, Lemas V, Yu AL, Vaughan J, Rivier J, Vale W 1987 Importance of FSH-releasing protein and inhibin in erythrodifferentiation. Nature 330:765-767 15. Thomsen G, Woolf T, Whitman M, Sokol S, Vaughan J, Vale W, Melton DA 1990 Activins are expressed early in Xenopus embroygenesis and can induce axial mesoderm and anterior structures. Cell 63:485-493 16. LaPolt PS, Soto D, Su J-G, Campen CA, Vaughan J, Vale W, Hsueh A 1989 Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells. Mol Endocrinol 3:1666-1673 17. Woodruff T, Meunier H, Jones P, Hsueh A, Mayo K 1987 Rat inhibin: molecular cloning of a- and /3-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 1:561-568 18. Esch FS, Shimasaki S, Cooksey K, Mercado M, Mason AJ, Ying SY, Ueno N, Ling N 1987 Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinology 1:388-396 19. Meunier H, Rivier C, Evans R, Vale W 1988 Gonadal and extragonadal expression of inhibin a, /3A and j8B subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247251 20. Roberts V, Meunier H, Vaughan J, Rivier J, Rivier C, Vale W, Sawchenko P 1989 Production and regulation of inhibin subunits in pituitary gonadotropes. Endocrinology 124:552-554 21. Sporn MB, Roberts AB, Wakefield LM, Assoian RK 1986 Transforming growth factor-/?: biological function and chemical structure. Science 233:532-534
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22. Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung A, Ninfa EG, Frey AT, Gash DJ, Chow EP, Fisher RA, Bertonis JM, Torres G, Wallner BP, Ramachandran KL, Ragin RC, Manganaro TF, MacLaughlin DT, Donahoe PK 1986 Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human genes in animal cells. Cell 45:685-698 23. Weeks D, Melton D 1987 A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF/3. Cell 51:861-867 24. Padgett R, St Johnson R, Gelbart W 1987 A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-/? family. Nature 325:81-84 25. Wozney J, Rosen V, Celeste A, Mitsock L, Whitters M, Kriz R, Hewick R, Wang E 1988 Novel regulators of bone formation: molecular clones and activities. Science 242:1528-1534 26. Attardi B, Miklos J 1990 Rapid stimulatory effect of activin-A on messenger RNA encoding the follicle-stimulating hormone /3-subunit in rat pituitary cell cultures. Mol Endocrinol 4:721-726 27. Vale W, Vaughan J, Yamamoto G, Bruhn T, Douglas C, Dalton D, Rivier C, Rivier J 1983 Assay of corticotropin releasing factor. Methods Enzymol 103:565-577 28. Schwall RH, Nikolics K, Szony E, Gorman C, Mason AJ 1988 Recombinant expression and characterization of human activin A. Mol Endocrinol 2:1237-1242 29. Gharib SD, Bowers SM, Need LR, Chin WW 1986 Regulation of rat luteinizing hormone subunit messenger ribonucleic acids by gonadal steroid hormones. J Clin Invest 77:582-589 30. Gharib SD, Wierman ME, Badger TM, Chin WW 1987 Sex steroid hormone regulation of follicle-stimulating hormone subunit messenger ribonucleic acid (mRNA) levels in the rat. J Clin Invest 80:294-299 31. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction fragments to high specific activity. Anal Biochem 132:6— 13 32. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5205 33. Bruning JL, Kintz BL 1977 Computational Handbook of Statistics. Scott, Foresman and Co, Dallas 34. Shaw G, Kamen R 1986 A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659-667 35. Malter JS 1989 Identification of an AUUUA-specific messenger RNA binding protein. Science 246:664-666 36. Casey JL, Hentz MW, Koeller DM, Caughman SW, Rouault TA, Klausner RD, Harford JB 1988. Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation. Science 240:924-928 37. Brock ML, Shapiro DJ 1983 Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell 34:207-214 38. Eisenstein RS, Rosen JM 1988 Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level. Mol Cell Biol 8:3183-3190 39. Krane I, Spindel ER, Chin WW 1991 Thyroid hormone decreases the stability and the poly A tract length of rat thyrotropin /3 subunit mRNA. Mol Endocrinol 5:469-475 40. Paul SJ, Ortolano GA, Haisenleder DJ, Stewart JM, Shupnik MA, Marshall JC 1990 Gonadotropin subunit disappearance after blockade of GnRH action: testosterone prolongs the availability of FSH/3 subunit mRNA. Mol Endocrinol 4:1943-1955
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