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Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Jean-François Ethier, Ph.D., Ottawa Regional Cancer Centre, Centre for Cancer Therapeutics, Third Floor, 503 Smyth Road, Ottawa, Ontario, K1H 1C4, Canada. E-mail: jfethier{at}uottawa.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The current model of activin signaling mechanism involves two types of transmembrane receptors (activin type I and type II receptors), which bind activin and transduce its signal into the cell (1, 4). Inhibins are well known for their opposing actions of activin activities (4). It has been proposed that inhibin competes with activin for the binding of the type II receptor and blocks the formation of an active activin receptor complex with the type I receptor, thereby uncoupling the downstream activin signaling pathways (5, 6, 7). However, evidence for this hypothesis was incomplete because inhibin has approximately a 10-fold lower affinity for the activin type II receptors compared with activin (8), which is inconsistent with the potent inhibin antagonism observed for most activin actions. In addition, some activin actions mediated through the activin type II receptors are not antagonized by inhibin (9, 10). These observations suggested that additional components, such as specific inhibin coreceptors, are involved in the inhibin mechanism of action. Accordingly, several inhibin-binding proteins have recently been identified (11, 12) and even characterized, such as the inhibin binding protein (InhBP) (13, 14). A major breakthrough in the elucidation of the inhibin mechanism was the recent demonstration that betaglycan binds inhibin and increases its affinity for the activin type II receptor (15), thereby enhancing the ability of inhibin to antagonize the activin signal.
Betaglycan is also known as the TGFß type III receptor, and it binds TGFß2 with higher affinity than TGFß1 or TGFß3 (16). It has been suggested that betaglycan is an enhancer of TGFß access to its own signaling type II receptor (17). Two regions of the extracellular domain of betaglycan are involved in this process: the endoglin-related domain located at the amino-terminal region (18) and the uromodulin-related domain located next to the transmembrane domain (19). Deletion studies have demonstrated that betaglycan binds inhibin through the uromodulin-related domain (16).
Because betaglycan can be part of both TGFß and inhibin receptor complexes, we hypothesized that TGFß might compete with inhibin for betaglycan binding, thereby blocking high-affinity inhibin binding to the activin type II receptors, which in turn would allow activin to signal more freely. In the present study, we examined whether TGFß1 and TGFß2 modulate inhibin A activity through competition for betaglycan binding in pituitary gonadotropes, the classical targets of inhibin actions. We used the LßT2 gonadotrope cell line (20), which is activin responsive, as demonstrated by the increase of FSH secretion (21) and by the activation of FSHß-subunit and GnRHR promoter constructs in response to activin A (22).
Our results suggest that TGFß1 and TGFß2 may modulate inhibin activity by decreasing the number of available betaglycan molecules essential for inhibin antagonism of activin activity. This is the first study showing the functional significance underlying the dual ability of betaglycan to bind both TGFßs and inhibin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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When LßT2 cells were transfected with either the oFSHß promoter or the GRAS promoter, each was activated by activin A in a dose-dependent manner (Fig. 1
). The oFSHß promoter was stimulated up to 2-fold, whereas the GRAS element was stimulated up to 5-fold by activin A. The calculated EC50 values were 166 pM and 39 pM, respectively. Where applicable, subsequent experiments were performed using 0.5 nM activin A for near-maximal activity of both promoters.
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, inhibin A antagonized activin A stimulation of both the oFSHß promoter and the GRAS promoter in a dose-dependent manner (IC50 = 0.13 nM and 0.17 nM, respectively). For subsequent experiments involving inhibin A, a dose of 0.5 nM was used.
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). These effects were first significant at 0.4 nM TGFß1 and 4 nM TGFß2.
Because TGFß and activin can trigger the same signaling pathway and generate similar responses (25), we examined whether the increases in the promoter activities were the result of their direct stimulation by TGFß. LßT2 cells transfected with either construct were treated with increasing concentrations of TGFß1 or TGFß2 alone (
4 nM), but no stimulation of either the oFSHß or GRAS promoter was observed (Fig. 4). Another possible explanation for the increase of the promoters’ activities by TGFß reported in Fig. 3 would be that TGFßs augment the sensitivity of the cells to activin A by increasing, for example, the number of activin receptors at the cell surface or by up-regulating intracellular components of the activin signaling pathway. To test these possibilities, the cells were coincubated with activin A and increasing concentrations of TGFß1 or TGFß2 in the absence of inhibin A. Even at high concentrations, neither TGFß1 nor TGFß2 augmented either activin-induced promoter activity (Fig. 5). In contrast, both TGFß1 and TGFß2 stimulated the TGFß-responsive reporter construct p3TP-lux when transfected into Chinese hamster ovary (CHO) cells, confirming the bioactivity of the TGFß preparations used in these experiments (Fig. 6). Basal p3TP-lux activity in LßT2 cells was low and was only marginally increased by TGFßs (data not shown).
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shows that LßT2 cells express the activin type II and type IIB receptors, InhBP and betaglycan mRNAs, indicating that the necessary receptor components required for inhibin and activin activities are present in this cell type. Because betaglycan is known to bind to both TGFßs and inhibin, we next addressed the question whether TGFß can compete with inhibin for binding to its coreceptor. The ability of inhibin A, TGFß1, and TGFß2 to compete for radiolabeled inhibin A binding to the LßT2 cells was determined by competition studies. As shown in Fig. 8, unlabeled inhibin A competed with radiolabeled inhibin A for 75% of the total binding with an IC50 of 90 pM. Both TGFß1 and TGFß2 also competed for a portion of inhibin binding to the LßT2 cells (25% and 40% of the total binding, respectively). TGFß2 had an IC50 of 72 pM, similar to that of inhibin A, whereas TGFß1 was less potent (IC50 = 280 pM).
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, lane 1). This binding was competed by excess unlabeled inhibin A, showing the reversibility of the interactions (Fig. 9, lane 2). Activin A did not compete with inhibin, suggesting that these binding complexes are inhibin specific (Fig. 9, lane 3). Most importantly, TGFß2 competed with inhibin for binding to the high molecular mass proteins (145- and >200-kDa complexes), but it was less efficient in competing for the smaller molecular mass protein species (Fig. 9, lane 4), suggesting that it competes specifically with inhibin for the binding to proteins consistent with the size of betaglycan core protein and its glycosylated forms. TGFß1 also competed with inhibin for the binding to these high molecular mass proteins (data not shown).
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). Unlabeled inhibin A competed with radiolabeled inhibin for the binding to these betaglycan-related species (Fig. 10, lane 2), whereas activin A had no effect (Fig. 10, lane 1). Importantly, the presence of TGFß1 (Fig. 10, lane 3) or TGFß2 (Fig. 10, lane 5) during the affinity labeling procedure almost completely abolished the immunoprecipitation of these affinity-labeled inhibin-binding species, which is consistent with the cross-linking data shown in Fig. 9.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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LßT2 cells were shown to express betaglycan mRNA, which is consistent with the potent activity of inhibin A observed in these cells given the current model of inhibin action (11). We have also shown that inhibin A binds to betaglycan in LßT2 cells. Betaglycan is a transmembrane proteoglycan containing heparan and chondroitin sulfate chains. Betaglycan appears at the cell surface either devoid of glycosaminoglycan chains (core protein = 115 kDa) or in various glycosylated forms (>200 kDa). The glycosaminoglycan chains are neither necessary for the interaction of betaglycan with inhibin, nor do they impede this interaction (16), so inhibin can bind to both the nonglycosylated and the glycosylated forms of betaglycan.
Competition studies showed that TGFß1 and TGFß2 competed with inhibin A for the binding to LßT2 cells, suggesting that TGFß may interfere with inhibin for the binding to its membrane receptors. Using an immunoprecipitation approach, we identified betaglycan as one of the inhibin binding proteins competed by TGFß1 and TGFß2. Taken together, our results suggest that TGFß1 and TGFß2 compete with inhibin for the binding of betaglycan. This should result in a decrease of inhibin potency to antagonize activin. This competition thus can explain our observation that TGFßs rescued the activin A-induced promoter activities when inhibin A was present.
We have found that TGFß2 is a more effective competitor than TGFß1 for the binding of inhibin to the LßT2 cells. Previous studies using COS-1 cells expressing deletion mutants of betaglycan containing either of the TGFß-binding regions have determined that inhibin binds to the uromodulin-related region of betaglycan (16). Consistent with our results, those studies showed that TGFß2 is a more effective competitor than TGFß1 for inhibin binding to the uromodulin-related region.
Our studies here extend the findings of Esparza-Lopez et al. (16) by providing the functional significance for the ability of betaglycan to bind both TGFß and inhibin. TGFß1 and TGFß2 each rescued the activin-induced activity of both the FSHß promoter and the GRAS promoter in LßT2 cells when inhibin A was present. For the FSHß promoter, TGFß2 is more potent than TGFß1 in completely reversing inhibin suppression of activin stimulation. However, a more complex pattern has been observed for the GRAS promoter, whereby TGFß1 has a similar or higher potency than TGFß2, but reversal of the inhibin-suppressed GRAS promoter activity was only partial in two experiments (e.g. Fig. 3B) even with 40 nM TGFß2. This discrepancy is not understood.
TGFß1 and especially TGFß2 are effective competitors of inhibin binding to the LßT2 cells with an IC50 of 280 and 72 pM, respectively. However, the TGFß concentrations needed to reduce the inhibin activity by 50% is much higher (0.4–4 nM). This apparent discrepancy is due to the different doses of inhibin used in these experiments. The competition studies (Fig. 8) were performed at room temperature during 4 h with a lower inhibin concentration (<250 pM inhibin A tracer) compared with 0.5 nM inhibin A treatment over 24 h at 37 C. Correspondingly, a lower dose of TGFß was necessary to block 50% of the inhibin binding to the LßT2 cells, resulting in a lower IC50 for the competition studies than for the transcriptional studies.
The present study used a gonadotrope cell line and activin-sensitive promoters transcriptionally active in gonadotropes. The physiological significance of the findings in the pituitary is not yet known. However, it is believed that gonadal inhibin acts on the pituitary in an endocrine manner, whereas activin and TGFß have autocrine and paracrine actions in the pituitary (26, 27). The local concentration of TGFß in the pituitary may therefore be sufficient to counteract the inhibin concentrations that reach the gland. Although lactotrope cells are the main target of TGFß actions in the anterior pituitary (27), we can speculate that TGFß may have an important role in the balance between negative and positive factors determining the net activin biological activity on gonadotropes. However, further studies are needed to confirm the effects of TGFß on activin-induced activity of the endogenous GnRHR and FSHß promoters in gonadotropes.
It is currently unknown whether TGFß can modulate the activity of inhibin B, but the recent identification of the inhibin B-specific binding protein, InhBP or p120 (13, 14), suggests that inhibin B and inhibin A may use different mechanisms of action. Accordingly, TGFß might not modulate some of the biological actions of inhibin B mediated by InhBP.
One should keep in mind that the biologically relevant form of activin in the pituitary is probably activin B because only the ßB-subunit is detected in the pituitary, at least in the rat (28). Our results were generated using activin A because activin B was not available at the time of the study. Further studies need to be done to determine whether TGFßs and inhibins can similarly modulate activin B activity.
In conclusion, betaglycan appears to play a pivotal role in some inhibin actions and, by inference, in some activin actions. Because TGFßs, activin, and inhibin can be present in the same spatio-temporal location, it is possible that TGFß may facilitate the actions of activin by preventing inhibin binding to betaglycan and thereby reducing its effectiveness in antagonizing activin. This model could have applications in several tissues including the gonads, placenta, pituitary, and bone.
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MATERIALS AND METHODS |
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and ßA cDNAs (29). More than 99% purity was confirmed by SDS-PAGE under nonreducing and reducing conditions, and by bioassay and activin A ELISA. Purified rh-inhibin-A was obtained from Biotech Australia (Roseville, New South Wales, Australia). rh-TGFß1 was purchased from PreproTech (Rocky Hill, NJ) and rh-TGFß2 was from Sigma-Aldrich Corp. (St. Louis, MO). The reporter construct pGL3.5–5oFSHß was a gift from William Miller (Department of Biochemistry, North Carolina State University, Raleigh, NC). The reporter construct 3XGRAS-PRL-lux was a gift from Buffy S. Ellsworth (Colorado State University, Ft. Collins, CO). p3TP-lux reporter plasmid was obtained from Joan Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY). The pCMVß vector was from CLONTECH Laboratories, Inc. (Palo Alto, CA). All cell media and fetal bovine serum (FBS) were obtained from Trace Biosciences (Melbourne, Victoria, Australia). The LßT2 mouse cell line was generously given by Pamela Mellon (University of California, San Diego, CA). The protein G agarose, the avian myeloblastosis reverse transcriptase, and Fugene6 transfecting reagent were from Roche Diagnostics Australia (Nunawading, Victoria, Australia). The luciferin was from Promega Corp. (Madison, WI). Galacton-Star galactosidase (Gal) substrate was from Tropix (Bedford, MA). The RNeasy kit was from QIAGEN Pty Ltd. (Clifton Hill, Victoria, Australia). The DNA-free kit was from Ambion, Inc. (Austin, TX). The bis(sulfosuccinimidyl)suberate (BS) (3) was from Pierce Chemical Co. (Rockford, IL). Octyl-ß-D-glucopyranoside was from Sigma-Aldrich Corp. The affinity-purified antibody directed against the extracellular domain of the human betaglycan was obtained from R&D Systems (Minneapolis, MN).
Transfection of LßT2 Cells
LßT2 cells were maintained in DMEM, buffered with bicarbonate and supplemented with 10% FBS, and were cultured at 37 C in a 5% CO2 environment. For transient transfection of the reporter constructs, 500,000 cells per well were cultured in 24-well plates (70–80% confluence) for 24 h. The Fugene6 reagent was then used for transfections at a ratio of 1:3 (micrograms of DNA to microliters of Fugene6 reagent) according to the manufacturer’s instruction. Cells were transfected with 250 ng reporter construct and 25 ng pCMVß vector to monitor transfection efficiencies. Each treatment was applied to triplicate cultures 24 h post transfection; the cells were washed with PBS and the medium was changed to DMEM and 0.2% FBS with the appropriate concentration of activin A, inhibin A, TGFß1, and TGFß2. The cells were then incubated for a further 24 h before assay. In some experiments, transient transfections were also performed on cells cultured on 48-well plates using 125 ng reporter construct and 12.5 ng pCMVß.
Luciferase and ß-Gal Assays
Cells were washed twice with ice-cold PBS and then lysed in 200 µl lysis buffer (1% Triton X-100, 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol). The cells were then incubated on ice for 30 min before collection of the cell lysate. For the luciferase assay, 50 µl of cell lysate were mixed with 300 µl of assay buffer [25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 15 mM potassium phosphate buffer (pH 7.8), 1 mM dithiothreitol, 2 mM ATP]. The luciferase activity was measured for 2 sec using a Berthold luminometer (Berthold Australia, Bundoora, Victoria, Australia) after injection of the luciferase substrate. For the ß-Gal assay, 10 µl of supernatant was mixed with 50 µl of Galacton-Star galactosidase substrate, and the ß-Gal activity was counted after a 30-min incubation using a LumiCount 96-well plate reader (Packard, Meriden, CT). The luciferase activities are represented as relative activities (luciferase activity divided by the matching ß-Gal activity).
RT-PCR
Total RNA was extracted from 3 x 106 LßT2 cells using the RNeasy kit, followed by a deoxyribonuclease treatment to remove any genomic DNA contaminant. Five hundred nanograms of total RNA were then reverse transcribed using 100 ng random hexamers and 1.6 U avian myeloblastosis reverse transcriptase in a 30-µl-volume reaction. One microliter of the cDNA solution was then subjected to PCR amplification using primers specific for mouse activin receptor II, activin receptor IIB, InhBP, or betaglycan sequences in an OmniGene thermal cycler (Hybaid, Teddington, UK). The amplified fragments were then separated on a 1.5% agarose gel and visualized with ethidium bromide staining.
Competition for [125I]Inhibin Binding to LßT2 Cells
For binding studies, LßT2 cells were plated at 250,000 cells per well in 48-well plates. After 24 h, medium was changed to DMEM containing 0.6% BSA, insulin (0.1 µg/ml), and transferrin (0.5 µg/ml). One day later, the cells were washed and then incubated in 50 mM HEPES-buffered DMEM containing 0.1% BSA and protease inhibitors (0.4 mM EDTA and 100 µg/ml phenylmethylsulfonylfluoride). Iodinated inhibin A was prepared as previously described (12). Binding affinity was assessed by incubating cells for 4 h at 23 C on an orbital mixer with [125I]inhibin A (50,000 cpm/0.125-ml/well, corresponding to a final concentration of 150–300 pM) in the absence or presence of unlabeled inhibin A, TGFß1, or TGFß2 (each added at 11 concentrations in singlicate). Nonspecific binding, identified as binding that was not competed at high concentrations of unlabeled inhibin A (>20 nM), was subtracted from all binding data. The binding reaction was terminated by placing the culture plates on ice and washing the cells three times with ice-cold PBS. Cells were lysed in 0.1 ml 0.1% Triton X-100 in PBS for 15 min at room temperature, and radioactivity recovered from each well was counted in a 
-counter.
Affinity Cross-Linking
Cells for affinity labeling studies were handled similarly to those for binding studies, except that 2 x 106 cells/4 ml medium/well were initially plated in 12-well plates. On d 2, LßT2 cells were incubated for 4 h at room temperature in 0.45 ml binding medium containing 200–400 pM [125I]inhibin A with or without 10 nM unlabeled inhibin A, activin A, TGFß1, or TGFß2. Cell monolayers were washed three times with ice-cold cross-linking buffer (50 mM HEPES, 125 mM NaCl, 5 mM KCl, 5 mM MgSO4, 1.2 mM CaCl2, pH 7.4), and then they were incubated with 0.25 mM BS (3) in cross-linking buffer for 30 min at 4 C. The cells were washed twice with quenching buffer (85 mM Tris, 30 mM NaCl, pH 7.8), after which each monolayer was lysed with 0.1 ml 1% octyl-ß-D-glucopyranoside in quenching buffer containing 4 mM EDTA and 500 µg/ml phenylmethylsulfonylfluoride. The supernatant was concentrated by evaporation in a Speed-Vac instrument (Savant Instruments, Farmingdale, NY). The cross-linked proteins were then separated by 7.5% SDS-PAGE under nonreducing conditions. Gels were dried and [125I]inhibin-binding protein complexes were visualized by autoradiography using BioMax film (Eastman Kodak Co., Rochester, NY).
Immunoprecipitation of Inhibin Affinity-Labeled Complexes
Affinity-labeled LßT2 cell lysate (0.1 ml) was diluted 1:4 with quenching buffer, after which 2 µg antiserum directed against betaglycan were added, and the mixture was incubated overnight at 4 C with mixing. Immune complexes were precipitated by the addition of 10 µl protein G agarose, incubation for 2 h at room temperature, and then centrifugation. The pellets were washed twice with 0.5 ml RIPA buffer (50 mM Tris, pH 8; 150 mM NaCl; 10 mM EDTA; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate). The bound proteins were eluted by boiling in 40 µl SDS-PAGE loading buffer and then separated by 7.5% nonreducing SDS-PAGE and visualized by autoradiography.
Statistical Analyses
All luciferase values are given as the mean ± SD calculated from transfections performed in triplicate. The data are representative of three or more separate experiments. ANOVA was used for statistical analyses, and the values were subjected to a Fischer’s least significant difference test to evaluate differences between samples. P < 0.05 was considered significant. The dose-response curves were fitted using Prism software (version 2, GraphPad Software, Inc., San Diego, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: BS, Bis(sulfosuccinimidyl)suberate; CHO, Chinese hamster ovary; FBS, fetal bovine serum; Gal, galactosidase; GnRHR, GnRH receptor; GRAS, GnRH receptor-activating sequence; InhBP, inhibin binding protein; o, ovine; rh-, recombinant human.
Received for publication January 11, 2002. Accepted for publication August 27, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), TGFß1 (
), or TGFß2 (
) for Binding to LßT2 Cells
