Transforming Growth Factor-ß Modulates Inhibin A Bioactivity in the LßT2 Gonadotrope Cell Line by Competing for Binding to Betaglycan

Jean-François Ethier, Paul G. Farnworth, Jock K. Findlay and Guck T. Ooi

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activin stimulates expression of GnRH receptor (GnRHR) and FSH ß-subunit in gonadotropes. Inhibin antagonizes activin actions on the gonadotropes, but its molecular mechanism of action remains poorly understood. It has been suggested that inhibin exerts its antagonistic effects by competing with activin for the binding of the activin receptor complex. Betaglycan has recently been identified as an inhibin-binding accessory protein in this process. Because both inhibin and TGFß bind betaglycan, we examined whether TGFß can modify inhibin’s antagonism of activin-induced transcription in gonadotrope cells. Two activin-responsive reporter constructs were used, the first containing 5.5 kb of the ovine FSHß promoter (oFSHßluc), and the second containing three copies of the activin-responsive sequence of the GnRHR promoter (3XGRAS-PRL-lux). These constructs were transfected into the gonadotrope cell line LßT2. The oFSHßluc and 3XGRAS-PRL-lux activities stimulated by 0.5 nM activin A were decreased by up to 50% in a dose-dependent manner by inhibin A. TGFß1 and TGFß2 (0–4 nM), alone or in the presence of activin A, did not significantly affect the promoter elements. However, with increasing doses of TGFß1 or TGFß2, inhibin A antagonism of activin A activity was partly or completely reversed. Competition studies with radiolabeled inhibin A showed that TGFß1 and TGFß2 competed with [125I]inhibin for the binding to LßT2 cells (IC50 = 280 pM and 72 pM, respectively). Immunoprecipitation studies of [125I]inhibin A cross-linked receptor complexes confirmed that TGFß1 and TGFß2 competed with inhibin A for the binding of betaglycan. These results suggest that TGFß competition with inhibin for binding to betaglycan interferes with inhibin’s suppression of activin-induced FSHß and GnRHR promoters in LßT2 cells. We propose that under certain circumstances, TGFß may facilitate activin biological activity by hindering the access of inhibin to its coreceptor betaglycan.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ACTIVIN AND INHIBIN were originally identified as potent modulators of FSH synthesis and secretion (1). It was further shown that they modulate many other pituitary functions, including the synthesis of the GnRH receptor (GnRHR) by gonadotropes (2, 3). Despite elucidation of these actions of activin and inhibin, very little is known about the actual mechanisms involved in the activin/inhibin regulation of gonadotrope-specific genes.

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effect of Inhibin A on Activin A-Induced Transcription of oFSHß and GRAS (GnRH Receptor Activating Sequence) Element Promoters
To assess inhibin bioactivity in LßT2 cells, we used two different activin-responsive promoter reporter constructs in transient transfection studies. The first construct contained 5.5 kb of the 5'-flanking region of the ovine FSHß (oFSHß) gene (23), and the second construct contained three repeats of the activin-responsive element GRAS of the murine GnRHR promoter (24).

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. 1Go). 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.



View larger version (14K):
[in this window]
[in a new window]
 
Figure 1. Activin A Concentration-Dependent Transcriptional Activation of the oFSHß Promoter (A) and the GRAS Element (B)

LßT2 cells were transiently transfected with either the oFSHß promoter/reporter or the GRAS-PRL-lux promoter/reporter constructs along with pCMVß as an internal control. Luciferase activity values were normalized to the ß-Gal activity of the internal control vector. Values are the mean ± SD of triplicate results from a representative experiment that was performed three times with similar results.

 
We next determined whether inhibin A can antagonize the activin A-induced transcriptional activity of the oFSHß and GRAS element promoters. As shown in Fig. 2Go, 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.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 2. Concentration-Dependent Effects of Inhibin A on Activin A-Induced Transcriptional Activity

LßT2 cells were transiently transfected with either the oFSHß promoter (panel A) the GRAS-PRL-lux reporter constructs along with pCMVß as an internal control (panel B). Cells received 0.5 nM activin A and increasing concentrations of inhibin A and were incubated for 24 h. Luciferase activity values were normalized to the ß-Gal activity of the internal control vector, pCMVß. Values are the mean ± SD of triplicates from a representative experiment that was performed three times with similar results.

 
Effect of TGFß1 and TGFß2 on Inhibin Activity
To examine the effect of TGFß on inhibin-modulated FSHß promoter activity, transfected cells were concurrently exposed to 0.5 nM activin A, 0.5 nM inhibin A, and increasing doses of TGFß1 or TGFß2. Inhibin A suppressed the activin A-induced oFSHß promoter activity by 25%. Inhibin suppression of this activity was completely reversed by both TGFß1 and TGFß2, with the first significant effects seen at 40 nM for TGFß1 and 0.4 nM TGFß2 (Fig. 3AGo).



View larger version (42K):
[in this window]
[in a new window]
 
Figure 3. Effects of TGFß1 and TGFß2 on Inhibin Antagonism of Activin Activity

LßT2 cells were transiently transfected with either the oFSHß promoter (panel A) or the GRAS-PRL-lux reporter constructs along with pCMVß as an internal control (panel B). Cells were incubated for 24 h in the presence of 0.5 nM activin A, with or without 0.5 nM inhibin A and increasing concentrations of either TGFß1 or TGFß2. Luciferase activity values were normalized to the ß-Gal activity of the internal control vector, pCMVß. The values for each treatment are expressed as the percentage of the activity in cells stimulated by activin alone (which is taken as 100%, indicated by the bold line across the graphs). The level of activity resulting from cotreatment with activin and inhibin is indicated by a dashed line across the graphs. Values are the mean ± SD of triplicates from a representative experiment performed three times. Values that were significantly different from the activin + inhibin treatment are indicated with one (P < 0.05) or two (P < 0.01) asterisks.

 
The same treatment regimen was also applied to LßT2 cells transfected with the GRAS promoter. Inhibin A reduced the activin A-induced GRAS promoter activity by 40%. Similar to what was observed with the oFSHß promoter, the suppression of GRAS promoter activity resulting from coincubation of activin A and inhibin A was significantly relieved when increasing doses of both TGFß1 and TGFß2 were added (Fig. 3BGo). 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. 4Go). Another possible explanation for the increase of the promoters’ activities by TGFß reported in Fig. 3Go 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. 5Go). 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. 6Go). Basal p3TP-lux activity in LßT2 cells was low and was only marginally increased by TGFßs (data not shown).



View larger version (17K):
[in this window]
[in a new window]
 
Figure 4. Effects of TGFß1 and TGFß2 Alone on the Transcriptional Activity of the oFSHß Promoter or the GRAS Element

LßT2 cells were transiently transfected with either the oFSHß promoter (panel A) or the GRAS-PRL-lux reporter constructs along with pCMVß as an internal control (panel B). Cells received increasing concentrations of TGFß1 or TGFß2 and were then incubated for 24 h. Luciferase activity values were normalized to the ß-Gal activity of the internal control vector, pCMVß. The values of each treatment are expressed relative to the activity in cells receiving no treatment, which was set to 1. Values are the mean ± SD of triplicates from a representative experiment performed three times. No statistically significant differences between treatments were detected.

 


View larger version (15K):
[in this window]
[in a new window]
 
Figure 5. TGFß1 and TGFß2 Do Not Increase the Sensitivity of LßT2 Cells to Activin

LßT2 cells were transiently transfected with either the oFSHß promoter (panel A) or the GRAS-PRL-lux reporter constructs along with pCMVß as an internal control (panel B). Cells were treated with 0.5 nM activin A and increasing concentrations of TGFß1 or TGFß2 and were incubated for 24 h. Luciferase activity values were normalized to the ß-Gal activity of the internal control vector, pCMVß. The values of each treatment are expressed relative to the activity in cells receiving no treatment, which was set to 1. Values are the mean ± SD of triplicates from a representative experiment performed three times. No statistically significant effects of treatments were detected.

 


View larger version (15K):
[in this window]
[in a new window]
 
Figure 6. TGFß1 and TGFß2 Are Potent Transcriptional Stimulators of p3TP-lux Reporter Activity in CHO Cells

CHO cells transiently transfected with p3TP-lux plasmids were treated with either buffer alone (Control), 0.5 nM activin-A, 0.4 nM TGFß1, or 0.4 nM TGFß2 for 24 h. Luciferase activity values were normalized to the ß-Gal activity of the internal control vector, pCMVß. Values are the average of duplicate measurements (±range) from a representative experiment.

 
TGFß Competition of Inhibin Binding
We verified by RT-PCR whether LßT2 cells express mRNAs encoding the currently known inhibin binding proteins. Figure 7Go 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. 8Go, 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).



View larger version (39K):
[in this window]
[in a new window]
 
Figure 7. Presence of Inhibin Binding Protein mRNA in LßT2 Cells

RT-PCR was performed on total RNA extracted from LßT2 cells for the detection of the mRNAs encoding the activin type II receptor (lanes 1 and 2), the activin type IIB receptor (lanes 3 and 4), InhBP (lanes 5 and 6), and betaglycan (lanes 7 and 8). The expected sizes of the amplified fragments were 700, 403, 282, and 697 bp, respectively. For each set of amplifications, a negative control was included where the reverse transcription (RT) was omitted from the reaction (lanes 1, 3, 5, and 7). Molecular weight markers were run in the final lane (MW, lane 9).

 


View larger version (15K):
[in this window]
[in a new window]
 
Figure 8. Competition between [125I]inhibin A and Unlabeled Inhibin A ({bullet}), TGFß1 ({blacktriangleup}), or TGFß2 ({square}) for Binding to LßT2 Cells

[125I]Inhibin A was incubated with LßT2 cells in the presence of increasing concentrations of each competitor for 4 h at room temperature. The respective IC50 values indicated on the right were determined using Prism software.

 
Characterization of Inhibin A Binding Proteins Competed by TGFßs
To determine which inhibin binding proteins are competed by TGFß, radiolabeled [125I]inhibin A was cross-linked to the LßT2 cell membrane binding proteins in the absence or presence of 10 nM competitors (activin A, inhibin A, TGFß1, and TGFß2). This concentration of competitor was chosen to provide a range of 10- to 100-fold molar excess over the inhibin tracer used, the concentration of which was below 250 pM. [125I]Inhibin A bound to binding protein species consistent with the size of activin receptor II, betaglycan core protein, and glycosylated betaglycan (95-, 145-, and >200-kDa complexes, respectively; Fig. 9Go, lane 1). This binding was competed by excess unlabeled inhibin A, showing the reversibility of the interactions (Fig. 9Go, lane 2). Activin A did not compete with inhibin, suggesting that these binding complexes are inhibin specific (Fig. 9Go, 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. 9Go, 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).



View larger version (68K):
[in this window]
[in a new window]
 
Figure 9. TGFß Competition of Inhibin Binding

LßT2 cells were incubated with [125I]inhibin-A in the absence (lane 1) or presence (lane 2) of unlabeled inhibin A, activin A (lane 3), or TGFß2 (lane 4) (10 nM in each case). After washing, cells were treated with 0.25 mM BS (3 ) to cross-link [125I]inhibin to binding proteins. The cell lysate was separated on SDS-PAGE and analyzed by autoradiography. The relative molecular masses of the inhibin-binding protein complexes are indicated on the left.

 
To confirm that the high molecular mass inhibin binding proteins in LßT2 cells are betaglycan, the affinity-labeled complexes were immunoprecipitated with antibodies directed against betaglycan. Consistent with the results from Lewis et al. (15), the betaglycan antibody recognized the [125I]inhibin A cross-linked complexes with relative molecular masses higher than 200 kDa (Fig. 10Go). Unlabeled inhibin A competed with radiolabeled inhibin for the binding to these betaglycan-related species (Fig. 10Go, lane 2), whereas activin A had no effect (Fig. 10Go, lane 1). Importantly, the presence of TGFß1 (Fig. 10Go, lane 3) or TGFß2 (Fig. 10Go, 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. 9Go.



View larger version (64K):
[in this window]
[in a new window]
 
Figure 10. Characterization of Inhibin A-Binding Proteins from LßT2 Cells Competed by TGFß

[125I]Inhibin A was incubated with LßT2 cells in the absence (lane 4) or presence (lane 2) of 10 nM unlabeled inhibin A, activin A (lane 1), TGFß1 (lane 3), or TGFß2 (lane 5). The cells were then washed and cross-linked with 0.25 mM BS (3 ), and the cell lysates were immunoprecipitated with a betaglycan antibody. A cell sample was processed in absence of betaglycan antibody (lane 6). Immunoprecipitates were separated on SDS-PAGE and analyzed by autoradiography. The relative molecular mass of the inhibin binding species is indicated on the right. The lower molecular weight bands in lanes 1, 3, 4, and 5 represent free [125I]inhibin that was precipitated in the betaglycan-inhibin complex but that was not cross-linked.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The presence of activin in a tissue does not mean that it can generate a signal because many factors can alter activin biological activity. For example, the net activin biological activity is determined, in part, by extracellular factors, such as follistatin and inhibin, which oppose activin bioactivity. On the other hand, our results suggest that TGFß can also influence the net activin biological activity, but in a positive manner. We demonstrate, for the first time, that TGFß can modulate the bioactivity of inhibin A. LßT2 cells coincubated with TGFß1 or TGFß2, combined with inhibin A and activin A, had significantly higher activininduced transcriptional activities compared with cells incubated without TGFß. We showed that direct TGFß signaling to the oFSHß and GRAS promoters does not explain the increase of promoter activity when TGFß is incubated in the presence of activin A and inhibin A. In addition, neither TGFß1 nor TGFß2 increased the activin responsiveness of the LßT2 cells because coincubation of either TGFß1 or TGFß2 with activin does not result in an increase in transcriptional activity of either promoter compared with cells incubated with activin alone. Our results suggest that TGFßs use an alternative mechanism that potentiates activin activity in the presence of inhibin.

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. 3BGo) 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. 8Go) 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Recombinant human (rh-) activin A was purified to homogeneity as a side product from culture media obtained from mammalian cells stably transfected with the human inhibin {alpha} 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 {gamma}-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).


    ACKNOWLEDGMENTS
 
The authors are grateful to Dr. P. Mellon, who kindly provided the LßT2 cell line; to Drs. W. Miller, B. Ellsworth, and Joan Massagué for their respective gifts of the oFSHß, GnRHR, and p3TP-lux reporter constructs; Dr. P. Stanton for provision of purified activin A; and to Pauline Leembruggen and Ruth Escalona for excellent technical assistance.


    FOOTNOTES
 
This work was supported by National Health and Medical Research council of Australia (RegKeys 983212 and 198705) (to J.K.F.) and by Fonds pour la Formation de Chercheurs et l’Aide à la Recherche, Canada (to J.-F.E.).

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ethier JF, Findlay JK 2001 Roles of activin and its signal transduction mechanisms in reproductive tissues. Reproduction 121:667–675[Abstract/Free Full Text]
  2. Fernandez-Vazquez G, Kaiser UB, Albarracin CT, Chin WW 1996 Transcriptional activation of the gonadotropin-releasing hormone receptor gene by activin A. Mol Endocrinol 10:356–366[Abstract]
  3. Braden TD, Farnworth PG, Burger HG, Conn PM 1990 Regulation of the synthetic rate of gonadotropin-releasing hormone receptors in rat pituitary cell cultures by inhibin. Endocrinology 127:2387–2392[Abstract]
  4. Pangas SA, Woodruff TK 2000 Activin signal transduction pathways. Trends Endocrinol Metab 11:309–314[CrossRef][Medline]
  5. Lebrun JJ, Vale WW 1997 Activin and inhibin have antagonistic effects on ligand-dependent heteromerization of the type I and type II activin receptors and human erythroid differentiation. Mol Cell Biol 17:1682–1691[Abstract]
  6. Martens JW, de Winter JP, Timmerman MA, McLuskey A, van Schaik RH, Themmen AP, de Jong FH 1997 Inhibin interferes with activin signaling at the level of the activin receptor complex in Chinese hamster ovary cells. Endocrinology 138:2928–2936[Abstract/Free Full Text]
  7. Xu J, McKeehan K, Matsuzaki K, McKeehan WL 1995 Inhibin antagonizes inhibition of liver cell growth by activin by a dominant-negative mechanism. J Biol Chem 270:6308–6313[Abstract/Free Full Text]
  8. Mathews LS, Vale WW 1991 Expression cloning of an activin receptor, a predicted transmembrane serine kinase. Cell 65:973–982[Medline]
  9. Bilezikjian LM, Blount AL, Campen CA, Gonzalez-Manchon C, Vale W 1991 Activin-A inhibits proopiomelanocortin messenger RNA accumulation and adrenocorticotropin secretion of AtT20 cells. Mol Endocrinol 5:1389–1395[Abstract]
  10. Schubert D, Kimura H, LaCorbiere M, Vaughan J, Karr D, Fischer WH 1990 Activin is a nerve cell survival molecule. Nature 344:868–870[CrossRef][Medline]
  11. Harrison CA, Farnworth PG, Chan KL, Stanton PG, Ooi GT, Findlay JK, Robertson DM 2001 Identification of specific inhibin A-binding proteins on mouse Leydig (TM3) and Sertoli (TM4) cell lines. Endocrinology 142:1393–1402[Abstract/Free Full Text]
  12. Hertan R, Farnworth PG, Fitzsimmons KL, Robertson DM 1999 Identification of high affinity binding sites for inhibin on ovine pituitary cells in culture. Endocrinology 140:6–12[Abstract/Free Full Text]
  13. Chong H, Pangas SA, Bernard DJ, Wang E, Gitch J, Chen W, Draper LB, Cox ET, Woodruff TK 2000 Structure and expression of a membrane component of the inhibin receptor system. Endocrinology 141:2600–2607[Abstract/Free Full Text]
  14. Chapman SC, Woodruff TK 2001 Modulation of activin signal transduction by inhibin B and inhibin-binding protein (InhBP). Mol Endocrinol 15:668–679[Abstract/Free Full Text]
  15. Lewis KA, Gray PC, Blount AL, MacConell LA, Wiater E, Bilezikjian LM, Vale W 2000 Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. Nature 404:411–414[CrossRef][Medline]
  16. Esparza-Lopez J, Montiel JL, Vilchis-Landeros MM, Okadome T, Miyazono K, Lopez-Casillas F 2001 Ligand binding and functional properties of betaglycan, a co-receptor of the transforming growth factor-ß superfamily. Specialized binding regions for transforming growth factor-ß and inhibin A. J Biol Chem 276:14588–14596[Abstract/Free Full Text]
  17. Lopez-Casillas F, Wrana JL, Massague J 1993 Betaglycan presents ligand to the TGF ß signaling receptor. Cell 73:1435–1444[Medline]
  18. Lopez-Casillas F, Payne HM, Andres JL, Massague J 1994 Betaglycan can act as a dual modulator of TGF-ß access to signaling receptors: mapping of ligand binding and GAG attachment sites. J Cell Biol 124:557–568[Abstract]
  19. Pepin MC, Beauchemin M, Plamondon J, O’Connor-McCourt MD 1994 Mapping of the ligand binding domain of the transforming growth factor ß receptor type III by deletion mutagenesis. Proc Natl Acad Sci USA 91:6997–7001[Abstract]
  20. Alarid ET, Windle JJ, Whyte DB, Mellon PL 1996 Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development 122:3319–3329[Abstract/Free Full Text]
  21. Graham KE, Nusser KD, Low MJ 1999 LßT2 gonadotroph cells secrete follicle stimulating hormone (FSH) in response to activin A. J Endocrinol 162:R1–R5
  22. Pernasetti F, Vasilyev VV, Rosenberg SB, Bailey JS, Huang HJ, Miller WL, Mellon PL 2001 Cell-specific transcriptional regulation of follicle-stimulating hormone-ß by activin and gonadotropin-releasing hormone in the LßT2 pituitary gonadotrope cell model. Endocrinology 142:2284–2295[Abstract/Free Full Text]
  23. Huang HJ, Sebastian J, Strahl BD, Wu JC, Miller WL 2001 The promoter for the ovine follicle-stimulating hormone-ß gene (FSHß) confers FSHß-like expression on luciferase in transgenic mice: regulatory studies in vivo and in vitro. Endocrinology 142:2260–2266[Abstract/Free Full Text]
  24. Duval DL, Ellsworth BS, Clay CM 1999 Is gonadotrope expression of the gonadotropin releasing hormone receptor gene mediated by autocrine/paracrine stimulation of an activin response element? Endocrinology 140:1949–1952[Abstract/Free Full Text]
  25. Attisano L, Wrana JL 1998 Mads and Smads in TGF ß signalling. Curr Opin Cell Biol 10:188–194[CrossRef][Medline]
  26. Woodruff TK 1998 Regulation of cellular and system function by activin. Biochem Pharmacol 55:953–963[CrossRef][Medline]
  27. Sarkar DK, Pastorcic M, De A, Engel M, Moses H, Ghasemzadeh MB 1998 Role of transforming growth factor (TGF)-ß type I and TGF-ß type II receptors in the TGF-ß1-regulated gene expression in pituitary prolactin-secreting lactotropes. Endocrinology 139:3620–3628[Abstract/Free Full Text]
  28. Knight PG 1996 Roles of inhibins, activins, and follistatin in the female reproductive system. Front Neuroendocrinol 17:476–509[CrossRef][Medline]
  29. Tierney ML, Goss NH, Tomkins SM, Kerr DB, Pitt DE, Forage RG, Robertson DM, Hearn MT, de Kretser DM 1990 Physicochemical and biological characterization of recombinant human inhibin A. Endocrinology 126:3268–3270[Abstract]