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1.
J Endocrinol ; 176(1): 61-8, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12525250

ABSTRACT

Activin is a secreted growth factor that signals by binding two related classes of single transmembrane receptors at the cell surface. The interaction of activin with its receptors is highly regulated by other cell surface receptors, antagonistic ligands, and high affinity extracellular binding proteins such as follistatin. Two activin A mutants, the deletion mutant des[85-109]-activin A and the point mutant K102E-activin A (K102E), were investigated with respect to their ability to bind cell surface receptors and the binding protein follistatin. The deletion mutant exhibits low affinity for both receptors and follistatin whereas the point mutant fails to bind cell surface receptors but binds follistatin-288 with high affinity. K102E is able to compete with wild type activin to bind to follistatin and can thus increase the concentration of activin available for receptor binding and signaling. These findings underline the importance of the C-terminal region of activin for binding interactions and show that different residues in this region are involved in cell surface receptor and follistatin interactions.


Subject(s)
Activins/genetics , Activins/metabolism , Follistatin/metabolism , Inhibin-beta Subunits/genetics , Inhibin-beta Subunits/metabolism , Animals , Binding, Competitive , Biological Assay , Cell Line , Cells, Cultured , Follicle Stimulating Hormone/metabolism , Gene Deletion , Luciferases/metabolism , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/metabolism , Point Mutation , Protein Binding , Rats , Receptors, Cell Surface/metabolism
2.
Mol Cell Endocrinol ; 180(1-2): 47-53, 2001 Jun 30.
Article in English | MEDLINE | ID: mdl-11451571

ABSTRACT

Activin and inhibin research has provided important insight into reproductive physiology as well as many areas involving regulation of cell growth, differentiation and function. Progress in understanding the roles of these hormones in various cell and tissue types has been complimented by novel discoveries at the molecular level that have shed light on ligand/receptor interactions, signaling mechanisms and regulation. While the receptors and signaling pathway for activin are now well characterized, the molecular basis for inhibin action has remained relatively unclear. Here we summarize recent advances in understanding inhibin's mode of action focusing on our recent identification of betaglycan-glycan as an inhibin co-receptor capable of mediating inhibin action.


Subject(s)
Activins/antagonists & inhibitors , Inhibins/pharmacology , Receptors, Peptide/physiology , Activin Receptors , Activins/pharmacology , Activins/physiology , Animals , Humans , Inhibins/metabolism , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Proteoglycans/metabolism , Proteoglycans/physiology , Receptors, Peptide/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Receptors, Transforming Growth Factor beta/physiology
3.
Proc Natl Acad Sci U S A ; 98(13): 7570-5, 2001 Jun 19.
Article in English | MEDLINE | ID: mdl-11416224

ABSTRACT

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.


Subject(s)
Brain/metabolism , Corticotropin-Releasing Hormone/genetics , Corticotropin-Releasing Hormone/metabolism , Peptide Fragments/pharmacology , Receptors, Corticotropin-Releasing Hormone/metabolism , Amino Acid Sequence , Animals , CHO Cells , Corticotropin-Releasing Hormone/chemistry , Cricetinae , Cyclic AMP/metabolism , Genome, Human , Humans , Kinetics , Mice , Molecular Sequence Data , Organ Specificity , Peptide Fragments/chemical synthesis , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/physiology , Polymerase Chain Reaction , RNA, Messenger/genetics , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, Genetic , Transfection , Urocortins
4.
Clin Exp Pharmacol Physiol ; 28(3): 244-8, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11236135

ABSTRACT

1. The anterior pituitary is well documented to be under the control of central and peripheral factors that dynamically interact to affect cell-specific modulation of pituitary functions. However, it is becoming increasingly evident that these extrinsic factors work in concert with a variety of local products that exert autocrine/paracrine control on pituitary cells. 2. These factors modulate the activity of their target pituitary cells by altering the synthesis and secretion of cell-specific hormones and by exerting control on the growth and differentiation of cells of this tissue. Included in the list of growth factors and bioactive peptides known to be products of pituitary cells are the activins, possibly inhibins and follistatins. 3. These protein factors play an important role in the local modulation of several pituitary cell types and are crucial for the maintenance of normal follicle-stimulating hormone production and, thus, reproductive function and fertility.


Subject(s)
Glycoproteins/physiology , Growth Substances/physiology , Inhibins/physiology , Activins , Animals , Follistatin , Glycoproteins/pharmacology , Growth Substances/pharmacology , Humans , Inhibins/antagonists & inhibitors , Pituitary Gland/metabolism , Pituitary Gland/physiology
5.
Endocrinology ; 142(3): 1065-72, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11181520

ABSTRACT

Activins and transforming growth factor-beta (TGF beta) are crucial autocrine, paracrine, and endocrine modulators of anterior pituitary function. Activins regulate most pituitary cells and lactotropes are targets of TGF beta. Smad2 and Smad3 are two cellular mediators of activin/TGF beta signaling, whereas Smad7 is as an inducible, negative modulator of the pathway. This study was undertaken to evaluate Smad7 regulation in the pituitary. Activin A rapidly and transiently increased Smad7 messenger RNA (mRNA) levels of rat anterior pituitary (RAP), clonal gonadotrope (alpha T 3-1 and L beta T2), and corticotrope (AtT20) cells with an EC(50) of 0.1-0.2 nM. In RAP cells, activin A or TGF beta 1 had equivalent effects that were additive. Follistatin, known to bind and inactivate activins, prevented Smad7 induction by activin. Inhibin A partially antagonized activin A, perhaps reflecting gonadotrope-selective actions. This antagonism was also evident with alpha T 3-1 and L beta T2 gonadotropes. Forskolin had no measurable effect in RAP cells, but increased Smad7 mRNA levels in alpha T3-1 cells and decreased them in L beta T2 cells. Transient transfection of Smad7 along with 3TPLux, an activin/TGF beta-responsive reporter, blocked activin-mediated promoter activation in alpha T3-1 and AtT20 cells. In alpha T3-1 cells, which express endogenous follistatin mRNA, a follistatin-luciferase reporter, rFS(rin3)-Luc, was transcriptionally activated by activin A, or when cotransfected with a constitutively active ActRIB [Alk4(T>D)], Smad2, or Smad3. Smad7 blocked rFS(rin3)-Luc activation by activin A or Alk4(T>D). Together, these results point to a role of Smad7 in modulating activin/TGF beta signaling in the pituitary.


Subject(s)
DNA-Binding Proteins/physiology , Inhibins/physiology , Pituitary Gland, Anterior/physiology , Signal Transduction/physiology , Trans-Activators/physiology , Transforming Growth Factor beta/physiology , Activins , Animals , Cells, Cultured , DNA-Binding Proteins/genetics , Follistatin , Glycoproteins/pharmacology , Inhibins/pharmacology , Male , Mice , Osmolar Concentration , Pituitary Gland, Anterior/cytology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Smad7 Protein , Time Factors , Trans-Activators/genetics , Transcription, Genetic , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1
6.
Nature ; 404(6776): 411-4, 2000 Mar 23.
Article in English | MEDLINE | ID: mdl-10746731

ABSTRACT

Activins and inhibins, structurally related members of the TGF-beta superfamily of growth and differentiation factors, are mutually antagonistic regulators of reproductive and other functions. Activins bind specific type II receptor serine kinases (ActRII or IIB) to promote the recruitment and phosphorylation of the type I receptor serine kinase, ALK4 (refs 7-9), which then regulates gene expression by activating Smad proteins. Inhibins also bind type II activin receptors but do not recruit ALK4, providing a competitive model for the antagonism of activin by inhibin. Inhibins fail to antagonize activin in some tissues and cells, however, suggesting that additional components are required for inhibin action. Here we show that the type III TGF-beta receptor, betaglycan, can function as an inhibin co-receptor with ActRII. Betaglycan binds inhibin with high affinity and enhances binding in cells co-expressing ActRII and betaglycan. Inhibin also forms crosslinked complexes with both recombinant and endogenously expressed betaglycan and ActRII. Finally, betaglycan confers inhibin sensitivity to cell lines that otherwise respond poorly to this hormone. The ability of betaglycan to facilitate inhibin antagonism of activin provides a variation on the emerging roles of proteoglycans as co-receptors modulating ligand-receptor sensitivity, selectivity and function.


Subject(s)
Inhibins/metabolism , Proteoglycans/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction , Activin Receptors , Activin Receptors, Type II , Activins , Animals , Cell Line , Female , Humans , Inhibins/antagonists & inhibitors , Male , Mice , Ovary/metabolism , Protein Binding , Rats , Receptors, Growth Factor/metabolism , Receptors, Peptide/metabolism , Testis/metabolism
7.
Neuron ; 20(6): 1093-102, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9655498

ABSTRACT

Corticotropin releasing factor (CRF) is a major integrator of adaptive responses to stress. Two biochemically and pharmacologically distinct CRF receptor subtypes (CRFR1 and CRFR2) have been described. We have generated mice null for the CRFR1 gene to elucidate the specific developmental and physiological roles of CRF receptor mediated pathways. Behavioral analyses revealed that mice lacking CRFR1 displayed markedly reduced anxiety. Mutant mice also failed to exhibit the characteristic hormonal response to stress due to a disruption of the hypothalamic-pituitary-adrenal (HPA) axis. Homozygous mutant mice derived from crossing heterozygotes displayed low plasma corticosterone concentrations resulting from a marked agenesis of the zona fasciculata region of the adrenal gland. The offspring from homozygote crosses died within 48 hr after birth due to a pronounced lung dysplasia. The adrenal agenesis in mutant animals was attributed to insufficient adrenocorticotropic hormone (ACTH) production during the neonatal period and was rescued by ACTH replacement. These results suggest that CRFR1 plays an important role both in the development of a functional HPA axis and in mediating behavioral changes associated with anxiety.


Subject(s)
Anxiety/genetics , Gene Expression Regulation, Developmental/physiology , Neurosecretory Systems/growth & development , Receptors, Corticotropin-Releasing Hormone/genetics , Stress, Physiological/genetics , Adaptation, Physiological/physiology , Adrenal Gland Diseases/drug therapy , Adrenal Gland Diseases/genetics , Adrenal Gland Diseases/mortality , Adrenal Glands/growth & development , Adrenal Glands/pathology , Adrenocorticotropic Hormone/metabolism , Animals , Anxiety/metabolism , Behavior, Animal/physiology , Chimera , Corticosterone/pharmacology , Female , Homozygote , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Neurologic Mutants , Mutation/physiology , Neurosecretory Systems/pathology , Paraventricular Hypothalamic Nucleus/chemistry , Paraventricular Hypothalamic Nucleus/metabolism , Pituitary Gland/growth & development , Stress, Physiological/metabolism , Survival Analysis
8.
Endocrinology ; 139(7): 3361-4, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9645713

ABSTRACT

Activins and follistatins regulate all levels of the reproductive axis, including the pituitary where they stimulate and inhibit FSH production, respectively. Gonadotropes are known to express inhibin/activin betaB and activin-B (betaBbetaB) functions as an autocrine modulator of FSH production. By contrast, the mRNA for the activin-binding protein, follistatin, is present in most pituitary cells and folliculo-stellate cells may be the major source of the protein secreted by the anterior pituitary. Interleukin-1beta (IL-1beta) is one of several cytokines known to also influence the reproductive axis. IL-1beta inhibits the hypothalamo-pituitary-gonadal (HPG) axis by suppressing GnRH and gonadal steroid production. Because several pituitary cell types, including follistatin-producing folliculo-stellate cells, are targets of IL-1beta, cytokine effects on gonadotrope function were evaluated using cultured rat anterior pituitary cells. Activin-A (0.01 to 1 nM; 24h) increased basal FSH secretion approximately 2-fold. IL-1beta (0.005 to 0.5 nM) by itself had no effect on basal FSH secretion. However, IL-1beta attenuated FSH secretion in response to all concentrations of activin-A. These results suggest that the cytokine might stimulate the local production of a factor, such as follistatin, that antagonizes the action of activin-A. RNase protection analysis indicated that IL-1beta (0.005 to 5 nM) stimulated follistatin and inhibin/activin betaB mRNA accumulation in a time-dependent manner. These in vitro effects of IL-1beta were blocked by the specific IL-1 receptor antagonist (IL-lra) and were not mimicked by either rhIL-6 or lipopolysaccharide (LPS). Treatment of intact male rats with LPS (50 microg, i.v.), which increases plasma IL-1beta and induces IL-1beta expression in many tissues, including the pituitary, produced similar time-dependent increases in pituitary follistatin and inhibin/activin subunit mRNA levels. These results suggest that IL-1beta can modulate gonadotrope responses to activins by influencing the local balance of activin-B and follistatin within the pituitary.


Subject(s)
Follicle Stimulating Hormone/metabolism , Glycoproteins/genetics , Inhibins/genetics , Inhibins/pharmacology , Interleukin-1/physiology , Oligopeptides , Peptides/genetics , Pituitary Gland, Anterior/metabolism , RNA, Messenger/metabolism , Activins , Animals , Follistatin , Lipopolysaccharides/pharmacology , Male , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/drug effects , Rats , Rats, Sprague-Dawley
9.
Endocrinology ; 137(10): 4277-84, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8828487

ABSTRACT

Follistatins, activins, and inhibins are expressed in a wide range of tissues where they function as autocrine and/or paracrine factors. Activin B (beta B beta B) and inhibin B (alpha beta B) are the predominant forms expressed in the rat anterior pituitary. This study was designed to evaluate the regulation of the messenger RNAs (mRNAs) for inhibin alpha and beta B, and follistatin, relative to each other, using cultured rat anterior pituitary cells. Activin A stimulated follistatin (a maximal 4-fold stimulation by 6 h) and beta B (a maximal 1.7-fold stimulation after 2 h) mRNA levels. Although inhibin A dramatically decreased follistatin mRNA levels (34% of the control value after 24 h), it only marginally affected those of beta B (86% of the control value after 2 h). Follistatin inhibited the accumulation of its own mRNA (46% of the control value after 6 h), but had no statistically significant effect on beta B or alpha mRNA levels. Inhibin A was the only treatment that had an effect on alpha mRNA levels, causing a slight decrease (82% of the control value by 24 h). The effects of activin A and inhibin A on follistatin and beta B mRNA levels were dose dependent. Moreover, follistatin and inhibin A blocked the effects of activin A. Immunoneutralization experiments were performed to determine whether locally secreted activin B regulates the expression of these three mRNAs. A monoclonal antibody to activin B reduced follistatin and beta B mRNA levels (37% and 73% of the control value, respectively) and enhanced the stimulatory effect of exogenous activin A on these mRNAs (840% vs. 300% and 170% vs. 145% of the control value, respectively); there was no change in alpha mRNA accumulation. GnRH and activators of the protein kinase A (forskolin) and protein kinase C (12-O-tetradecanoylphorbol acetate) pathways also had differential effects on follistatin, beta B, and alpha mRNA levels. GnRH stimulated follistatin mRNA levels, but suppressed those of beta B. 12-O-Tetraphorbol acetate had no effect on beta B, but stimulated follistatin mRNA levels to the same extent as forskolin. Of these agents, only forskolin produced a marginal inhibitory effect on alpha mRNA accumulation. Testosterone decreased both follistatin and beta B mRNA levels without affecting those of alpha. The results of this study demonstrate that the local production of rat anterior pituitary follistatin, activin B, and inhibin B is regulated by hypothalamic, peripheral, and local factors in such a way that the ratios between activin B and its two inactivators, follistatin and inhibin B, are very tightly maintained.


Subject(s)
Glycoproteins/genetics , Hormones/physiology , Inhibins/genetics , Oligopeptides , Pituitary Gland/metabolism , RNA, Messenger/metabolism , Activins , Animals , Colforsin/pharmacology , Follistatin , Glycoproteins/pharmacology , Gonadotropin-Releasing Hormone/pharmacology , Inhibins/pharmacology , Male , Peptides/metabolism , Peptides/physiology , Rats , Rats, Sprague-Dawley , Tetradecanoylphorbol Acetate/pharmacology
10.
Endocrinology ; 137(1): 65-71, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8536643

ABSTRACT

Previous studies involving radioreceptor and functional assays have shown that CRF and glucocorticoids are able to modulate CRF receptors of the brain and anterior pituitary. In this study, we analyzed the effects of CRF, vasopressin (AVP), dexamethasone (DEX), and corticosterone on the regulation of CRF receptor (CRF-R1) messenger RNA (mRNA) levels in cultured rat anterior pituitary cells. CRF decreased CRF-R1 mRNA levels in a time- and concentration-dependent manner. In the presence of 10 nM CRF, CRF-R1 mRNA levels decreased within 1 h (to 65 +/- 3% of the control value; P < 0.01) with a maximal effect after 3 h (to 28 +/- 1% of the control value; P < 0.001). The concentration dependence of the inhibitory effect of CRF at 3 h correlated with that required for ACTH secretion (half-maximal at approximately 0.03 nM). Treatment with a maximal (100 nM) dose of AVP or a submaximal (0.1 nM) dose of CRF for 3 h reduced CRF-R1 mRNA levels to 66 +/- 3% and 53 +/- 6% of the control value, respectively. In the presence of both AVP and CRF, CRF-R1 mRNA levels were 32 +/- 3% of the control value. The incubation of cells for 3 h with 10 microM forskolin to activate adenylate cyclase or with 20 nM 12-0-tetradecanoylphorbol-13-acetate to activate protein kinase C resulted in a decrease in receptor mRNA levels to 40 +/- 9% (P < 0.01) and 28 +/- 8% (P < 0.001) of the control value, respectively, suggesting that the effects of CRF and AVP may be mediated by these pathways. DEX (20 nM) also caused a dose- and time-dependent decrease in mRNA levels. Maximal inhibition was observed after 3 h (to 31 +/- 6% of the control value; P < 0.001), with a partial recovery of mRNA levels at 24 or 48 h. Corticosterone similarly inhibited the accumulation of CRF-R1 mRNA in a dose- and time-dependent manner, but, in contrast to DEX, CRF-R1 mRNA levels returned almost to control levels after 24 h. These results indicate that the ability of CRF, AVP, and glucocorticoids to modulate the responses of corticotropes to CRF may be due in part to the actions of these agents on CRF-R1 mRNA accumulation.


Subject(s)
Corticotropin-Releasing Hormone/pharmacology , Glucocorticoids/pharmacology , Pituitary Gland, Anterior/metabolism , RNA, Messenger/metabolism , Receptors, Corticotropin-Releasing Hormone/genetics , Animals , Arginine Vasopressin/pharmacology , Base Sequence , Cells, Cultured , Colforsin/pharmacology , Male , Molecular Sequence Data , Oligonucleotide Probes/genetics , Pituitary Gland, Anterior/cytology , Rats , Rats, Sprague-Dawley , Tetradecanoylphorbol Acetate/pharmacology
11.
Proc Natl Acad Sci U S A ; 92(7): 2969-73, 1995 Mar 28.
Article in English | MEDLINE | ID: mdl-7708757

ABSTRACT

Corticotropin-releasing factor (CRF; corticoliberin) regulates the secretion of corticotropin (ACTH) and beta-endorphin and has a broad range of effects on the nervous, endocrine, reproductive, cardiovascular, gastrointestinal, and immune systems. Recently, human, rat, and mouse CRF receptors (CRF-R) have been cloned and functionally and anatomically characterized. We report here the cloning of a second CRF-R cDNA (CRF-RB), which encodes a protein of 431 amino acids, which is 16 amino acids longer and 68% similar to the previously cloned CRF-R, CRF-RA. When transiently expressed in COS-M6 cells, CRF-RB binds CRF with high affinity [Kd = 1.2 (0.57-2.5)nM] and transduces the CRF-stimulated signal of the accumulation of intracellular cAMP, which is inhibited by a CRF antagonist. Comparison of the amino acid sequences of CRF-RB and the previously cloned receptor reveals major differences in the N-terminal domain and in the extracellular loops, whereas the sequences of the intracellular loops are nearly identical. CRF-RB and related transcripts are expressed in the heart, as well as in other tissues, including the gastrointestinal tract, epididymis, and brain.


Subject(s)
Gene Expression , Myocardium/metabolism , Receptors, Corticotropin-Releasing Hormone/biosynthesis , Receptors, Corticotropin-Releasing Hormone/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Cell Line , Chlorocebus aethiops , Cloning, Molecular , Cyclic AMP/metabolism , DNA Primers , DNA, Complementary/metabolism , Duodenum/metabolism , In Situ Hybridization , Intestinal Mucosa/metabolism , Kidney , Kinetics , Male , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Rats , Receptors, Corticotropin-Releasing Hormone/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Testis/metabolism , Transfection
12.
Endocrinology ; 133(6): 2545-53, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8243276

ABSTRACT

The production of inhibin/activin by cultured rat anterior pituitary cells was evaluated using specific antisera to inhibin/activin alpha, beta A, and beta B subunit proteins (anti-alpha, anti-beta A, and anti-beta B). Cellular or secreted proteins recognized by the antisera were immunoprecipitated from metabolically labeled cells then analyzed by denaturing polyacrylamide gel electrophoresis. Immunoreactive inhibin/activin beta B proteins were visualized in both cell lysates and the media. Experiments with anti-beta B confirmed that activin-B (beta B beta B) is a local secretory product of cultured rat anterior pituitary cells. The secreted beta B-immunoreactive protein band had an apparent size of 24-25 kilodaltons (kDa) or 14-15 kDa, consistent with the size of unreduced beta B dimer or reduced monomer, respectively. Cell lysates contained two proteins that were specifically immunoprecipitated by anti-beta B. One of these had a mobility of greater than 95 kDa (unreduced) or 55-60 kDa (reduced), probably representing dimers or monomers of the beta B precursor, respectively. The second 14- to 15-kDa (reduced and unreduced) immunoreactive beta B protein band was verified to be the mature beta B monomer. Mature heterodimeric inhibin-B (alpha beta B) was not detected by either anti-alpha or anti-beta B. Multiple protein species, however, were observed to be specifically immunoprecipitated by incubation of cell lysates with anti-alpha. Mature beta A monomer was not detected in any of the samples. The regulation of cellular beta B production was monitored by evaluating its rate of synthesis in pulse-labeled cells. Treatment with either forskolin or 12-O-tetradecanoylphorbol acetate enhanced the rate of [35S]cysteine incorporation into the cellular 14- to 15-kDa beta B monomer, indicating that the activation of either protein kinase A or protein kinase C regulates its production. The rate of cellular beta B accumulation was also regulated by activin-A, inhibin-A, and follistatin; activin-A caused a 30% inhibition in contrast to the 70% stimulation by treatment with either inhibin-A or follistatin. Equimolar concentrations of activin-A and follistatin prevented the net effect produced by either factor alone. None of the immunoreactive alpha-forms was detectable under similar pulse-labeling conditions, and there was no apparent change in their level after labeling to equilibrium (up to 48 h). The observed changes in beta B accumulation may, therefore, reflect the regulated production of pituitary activin-B. Taken together, these results suggest that locally produced activin-B or gonadal activins exert an inhibitory tone on the production of pituitary activin-B and that this negative-feedback control is in turn modulated by inhibins and follistatins. The relative importance of pituitary and gonadal activins, inhibins, and follistatins in the proposed regulatory loop remains to be established.


Subject(s)
Inhibins/metabolism , Inhibins/physiology , Pituitary Gland, Anterior/metabolism , Animals , Cells, Cultured , Culture Media , Follicle Stimulating Hormone/metabolism , Immune Sera , Pituitary Gland, Anterior/cytology , Precipitin Tests , Rats
13.
Endocrinology ; 133(6): 2554-60, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8243277

ABSTRACT

The activin-binding protein, follistatin (FS), was immunoprecipitated from metabolically labeled rat anterior pituitary cells or their media using a specific antiserum to purified porcine FS (anti-FS). Several immunoreactive proteins, including one that had a mobility in the range of 42-44 kilodaltons (kDa), were detected in the cell lysates. When immunoprecipitates of the culture medium were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, a broad 35- to 46-kDa or 39- to 53-kDa band was visualized under unreducing or reducing conditions, respectively. Upon deglycosylation by treatment with N-glycosidase-F, the secreted product migrated as a sharp protein band with an apparent size of 35 kDa. The identity or the relatedness of the immunoprecipitated proteins to FS was verified by the ability of the C-terminally truncated form of recombinant human FS (rhFS288) to compete for binding to anti-FS. When the cultured rat anterior pituitary cells were treated with either forskolin or 12-O-tetradecanoylphorbol acetate, the accumulation of FS in the culture medium was stimulated by approximately 2.5-fold. These observations suggest that the activation of either the protein kinase A or the protein kinase C signaling pathway has a stimulatory effect on anterior pituitary FS production. A more dramatic stimulation of FS secretion (up to 7-fold) was observed when the rat anterior pituitary cells were treated with activin-A. The concentration dependence for this effect was within the same range that has been reported for most of the actions of activin-A. Inhibin-A suppressed basal FS secretion and blocked its stimulation by activin-A. To determine if locally produced FS exerts an influence on the response of gonadotropes to activins, the effects of anti-FS on FSH secretion were monitored. The ability of this FS antiserum to immunoneutralize the activity of FS was initially confirmed; anti-FS attenuated the inhibitory action of exogenous follistatin on FSH secretion. Treatment of cells with the antiserum increased the apparent sensitivity of gonadotropes to submaximal concentrations of activin-A. Moreover, the presence of the antiserum lowered the concentration of activin-A that was required to produce the maximum amount of FSH secretion, without changing the magnitude of the response. These results suggested that locally produced FS interferes with the secretory response of gonadotropes to activins. Changes in locally secreted FS may, therefore, represent a mechanism by which the response of rat anterior pituitary cells to incoming stimuli are tightly regulated.


Subject(s)
Glycoproteins/metabolism , Inhibins/physiology , Pituitary Gland, Anterior/metabolism , Activins , Animals , Cells, Cultured , Colforsin/pharmacology , Culture Media , Follistatin , Glycoproteins/physiology , Inhibins/pharmacology , Male , Pituitary Gland, Anterior/cytology , Rats , Tetradecanoylphorbol Acetate/pharmacology
14.
Biochem Biophys Res Commun ; 191(3): 1139-44, 1993 Mar 31.
Article in English | MEDLINE | ID: mdl-7916600

ABSTRACT

A cDNA for a GnRH receptor (mtGnRH-R) was obtained from a mouse gonadotropic pituitary cell line (alpha T3-1) by expression cloning. This full-length cDNA was subsequently used as a probe to clone a rat pituitary GnRH receptor (rGnRH-R). The two receptors differ by 13 amino acids and are 100% identical to those recently reported. The analysis of the cloned receptors by photoaffinity-labeling followed by SDS-PAGE reveals a major band of approximately 70 kDa. This is in contrast to the native rat pituitary and mouse alpha T3-1 receptors whose major labeled species migrate with an apparent size of approximately 45 kDa. Functional studies reveal that both receptors, when transiently expressed in COSM6 cells, can bind GnRH with high affinity and transduce the stimulation of IP3 accumulation in response to GnRH.


Subject(s)
Receptors, LHRH/physiology , Affinity Labels , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , Cloning, Molecular , Gene Expression , In Vitro Techniques , Inositol Phosphates/metabolism , Mice , Molecular Sequence Data , RNA, Messenger/genetics , Rats , Signal Transduction , Transfection
15.
Trends Endocrinol Metab ; 3(6): 218-23, 1992 Aug.
Article in English | MEDLINE | ID: mdl-18407103

ABSTRACT

Activins were initially isolated from gonadal fluids based on their ability to stimulate FSH secretion from gonadotropes of the anterior pituitary. They represent a group of homo/heterodimeric proteins that are structurally related to the transforming growth factor beta superfamily of growth and differentiation factors. The importance of activins as autocrine and paracrine modulators of a diverse set of biologic functions is now appreciated based on their widespread anatomic distribution and numerous biologic activities.

16.
Neuroendocrinology ; 54(4): 373-7, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1758579

ABSTRACT

The recent development of GnRH-secreting neuronal cell lines (GT1-1, GT1-3 and GT1-7 clones) has provided a model system for the study of the neural regulation of GnRH expression and secretion. We report here that activin-A stimulates GnRH secretion by GT1-7 cells in a dose-dependent manner, with an EC50 of approximately 2.5 ng/ml. The maximal response (50% stimulation) was achieved after 2 days of incubation with 20 ng/ml activin-A. Activin-A treatment increased total GnRH (secreted + cellular) in GT1-7 cells, possibly reflecting a stimulation of GnRH biosynthetic rates. The secretory effect of activin-A was also accompanied by a change in the cellular morphology to a more neuronal phenotype. The addition of TGF-beta (10 ng/ml), which is structurally related to activins, did not significantly increase secretion of GnRH by GT1-7 cells illustrating the specificity of the activin effect on this cell line. Although inhibin (20 ng/ml) alone did not directly affect the spontaneous secretion of GnRH, it was able to partially block the stimulatory effect of activin. The present study with the GT1-7 clonal cell line suggests that activin, and perhaps inhibin, might act at hypothalamic sites to regulate reproduction through the control of GnRH production and/or secretion.


Subject(s)
Gonadotropin-Releasing Hormone/metabolism , Inhibins/physiology , Neurons/metabolism , Transforming Growth Factor beta/physiology , Activins , Cell Line , Clone Cells/metabolism , Radioimmunoassay , Reproduction/physiology
17.
Mol Endocrinol ; 5(10): 1389-95, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1663577

ABSTRACT

The complexity of corticotropic cell regulation by multiple central and peripheral factors is well recognized. The present study provides evidence for the participation of an additional factor in the regulation of this cell type of the anterior pituitary. Using the clonal AtT20 cell line as a model for corticotropes, homodimeric activin-A was observed to suppress basal ACTH secretion and POMC mRNA accumulation by approximately 50%. These effects required prolonged treatment with activin-A and were concentration dependent; the half-maximum concentration was in the range of 30-50 pM. Consistently, AtT20 cells were found to express specific high affinity binding sites for [125I]activin-A. The simultaneous addition of inhibin-A along with increasing concentrations of activin-A did not alter the characteristics of the inhibition of ACTH secretion by activin-A alone. This is in contrast to observations with gonadotropes of the anterior pituitary as well as a number of other cell types in which inhibin-A can partially antagonize the biological actions of activin-A. The results may suggest the participation of a subclass of activin receptors that mediate effects on ACTH secretion and POMC mRNA accumulation. As previously shown, the incubation of AtT20 cells with a synthetic glucocorticoid, dexamethasone, attenuated basal ACTH secretion and POMC expression in a concentration-dependent manner. The inhibition of both of these parameters by activin-A, however, was independent of glucocorticoids, because the two agents were additive in their actions. In addition to effects on secretion and mRNA levels, treatment with activin-A also inhibited the rate of proliferation of AtT20 cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Adrenocorticotropic Hormone/metabolism , Inhibins/pharmacology , Pro-Opiomelanocortin/genetics , RNA, Messenger/genetics , Transcription, Genetic/drug effects , Activin Receptors , Activins , Animals , Cell Line , Gene Expression/drug effects , Inhibins/metabolism , Kinetics , Mice , Pituitary Gland, Anterior , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Receptors, Cell Surface/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Restriction Mapping
18.
Endocrinology ; 128(3): 1682-4, 1991 Mar.
Article in English | MEDLINE | ID: mdl-1900235

ABSTRACT

Activins, dimers of inhibin beta subunits, are potent stimulators of FSH secretion in vivo and in vitro and of FSH beta mRNA expression in rat anterior pituitary cultures. In this study, we investigated the possibility that locally secreted activin B (beta B beta B) may function as an autocrine modulator of basal FSH secretion and expression based on the previous observation that beta B is expressed within gonadotropes. The incubation of cultured rat anterior pituitary cells with a m mouse monoclonal antibody specific for the activin B homodimer (MAb-activin B) significantly attenuated the basal secretion of FSH in a concentration- and time-dependent manner, without influencing LH secretion. Moreover, MAb-activin B selectively inhibited FSH beta mRNA accumulation without affecting either LH beta or alpha subunit mRNAs. The MAb-activin B completely blocked the stimulation of FSH secretion by exogenous activin B, but not by activin A, confirming its specificity. As previously shown, inhibin A and follistatin significantly suppressed basal FSH secretion in these cultures. This inhibitory effect, albeit of lower magnitude, was still evident even in the presence of the MAb-activin B which by itself suppressed basal FSH secretion. These data suggest that the secretion of activin B by the gonadotropes of the anterior pituitary may serve as an autocrine signal in the selective modulation of FSH expression and secretion. Furthermore, the inhibitory actions of inhibins and follistatins on gonadotropes may, in part, be explained by their ability to interfere with the actions of endogenous activin B.


Subject(s)
Endocrine Glands/physiology , Oligopeptides , Peptides/physiology , Pituitary Gland, Anterior/physiology , Activins , Animals , Antibodies, Monoclonal , Cells, Cultured , Drug Interactions , Follicle Stimulating Hormone/antagonists & inhibitors , Follicle Stimulating Hormone/genetics , Follicle Stimulating Hormone/metabolism , Follistatin , Glycoproteins/pharmacology , Growth Hormone-Releasing Hormone/physiology , Inhibins/pharmacology , Luteinizing Hormone/genetics , Luteinizing Hormone/metabolism , Peptides/pharmacology , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/metabolism , RNA, Messenger/metabolism , Rats
19.
Mol Endocrinol ; 5(3): 347-55, 1991 Mar.
Article in English | MEDLINE | ID: mdl-1653891

ABSTRACT

We recently derived a GnRH-responsive pituitary cell line of the gonadotrope lineage (alpha T3-1) by targeted oncogenesis in transgenic mice. Here, we report studies characterizing the GnRH receptors present in these cells and the intracellular responses to GnRH treatment. The receptors in alpha T3-1 cells show specificity for different GnRH analogs, with dissociation constants very similar to those found in normal rat and mouse pituitary. The concentration of receptors is within the range found in normal pituitary. The addition of GnRH or GnRH agonists increases phosphoinositide turnover and protein kinase-C translocation to membranes, and enhances activation of voltage-sensitive calcium channels. However, GnRH does not affect cAMP levels. Analysis of alpha-subunit mRNA levels demonstrated induction by GnRH and phorbol esters. Our results indicate that GnRH initiates a cascade of intracellular events that generate a set of second messengers, one or more of which is involved in the regulation of gene expression. The responses of alpha T3-1 cells to GnRH appear to have characteristics equivalent to those of primary pituitary gonadotropes, indicating the utility of this cell line as a model system for the study of GnRH responses.


Subject(s)
Gonadotropin-Releasing Hormone/pharmacology , Receptors, LHRH/metabolism , Animals , Biological Transport , Calcium Channels/metabolism , Cell Line , Cells, Cultured , Clone Cells/drug effects , Clone Cells/metabolism , Cyclic AMP/metabolism , Mice , Phosphatidylinositols/metabolism , Protein Kinase C/metabolism , RNA, Messenger/metabolism , Receptors, LHRH/genetics , Triiodothyronine/pharmacology
20.
Am J Obstet Gynecol ; 163(4 Pt 1): 1307-12, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2171341

ABSTRACT

The present study evaluated the possible effect and mechanism of action of interleukin-1 beta in regulating the release of corticotropin-releasing factor and adrenocorticotropin hormone from human cultured placental cells. With the use of a primary monolayer culture of human placental cells at term, the addition of interleukin-1 beta increased the release of immunoreactive corticotropin-releasing factor with a dose- and time-dependent effect. The intracellular concentration of both cyclic adenosine monophosphate and cyclic guanosine monophosphate increased in the presence of interleukin-1 beta. The addition of indomethacin, a prostaglandin synthesis inhibitor, partially reversed the effect of interleukin-1 beta. The same doses of interleukin-1 beta stimulated the release of adrenocorticotropin hormone and this effect was partially reversed by the addition of a synthetic corticotropin-releasing factor antagonist or by indomethacin. This study showed that interleukin-1 beta increases the release of corticotropin-releasing factor and adrenocorticotropin hormone from cultured placental cells. This effect is associated with increased intracellular cyclic nucleotide concentrations and is in part reversed by a prostaglandin synthesis inhibitor.


Subject(s)
Adrenocorticotropic Hormone/metabolism , Corticotropin-Releasing Hormone/metabolism , Interleukin-1/pharmacology , Placenta/metabolism , Cells, Cultured , Corticotropin-Releasing Hormone/antagonists & inhibitors , Cyclic AMP/analysis , Cyclic GMP/analysis , Female , Humans , Indomethacin/pharmacology , Placenta/cytology , Placenta/drug effects , Pregnancy , Prostaglandins/physiology
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