©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibin Antagonizes Inhibition of Liver Cell Growth by Activin by a Dominant-negative Mechanism (*)

(Received for publication, November 29, 1994; and in revised form, January 17, 1995)

Jianming Xu (§) Kerstin McKeehan Koichi Matsuzaki Wallace L. McKeehan (¶)

From the Albert B. Alkek Institute of Biosciences and Technology, Department of Biochemistry and Biophysics, Texas A& University, Houston, Texas 77030-3303

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The beta:beta activin homodimer and alpha:beta inhibin heterodimer are mutual antagonists which share a common beta subunit. Recently, it has been shown that, similar to transforming growth factor-beta1, activin is an inhibitor of hepatocyte DNA synthesis. The activin receptor appears to be an obligatory complex of genetically distinct type I and II transmembrane serine/threonine kinases. Activin type I receptors, SKR1 and SKR2, were first cloned from well differentiated human hepatoma cells (HepG2). This prompted us to investigate the binding of activin and inhibin to receptors from HepG2 cells and the effect of the two ligands on DNA synthesis. Here we show that beta:beta activin binds to the activin type II receptor kinase (ActRII) which induces activin binding to the type I receptor kinase SKR2 to form ActRIIbulletbeta:betabulletSKR2 complexes in which an activin beta chain occupies each receptor subunit. Inhibin also binds to ActRII through its beta subunit, competes with the binding of activin to ActRII, but fails to form the ActRIIbulletSKR2 complex. No specific binding site for inhibin could be demonstrated in HepG2 cells. Inhibin, which had no activity of its own, antagonized the inhibitory effect of activin on DNA synthesis. The results suggest that inhibin may be a natural antagonist of assembly of the heterodimeric activin receptor complex through a dominant-negative mechanism.


INTRODUCTION

The activins and inhibins are dimeric cytokines with important roles in development and physiology(1) . Although first discovered as gonadal peptides that regulate spermatogenesis and oogenesis, both cytokines are widely expressed and cause diverse responses in cell types from both intragonadal and extragonadal tissues(1, 2) . Dependent on cell, tissue, and biological activity, activin or inhibin can act as a positive or negative effector, but both are generally antagonists of the other(1, 2, 3) . Activins are beta:beta homodimers while inhibins are alpha:beta heterodimers that share the beta subunit. Both are members of the transforming growth factor-beta (TGF-beta) (^1)superfamily of ligands whose activities are mediated by transmembrane serine/threonine kinase receptors(4, 5, 6, 7, 8) . Similar to TGF-beta1(9) , activin has recently been demonstrated to be an inhibitor of hepatocyte DNA synthesis (10) and to reduce liver mass by induction of hepatocyte apoptosis when administered in vivo(11) . In contrast to TGF-beta1, which is expressed in nonparenchymal cells after partial hepatectomy and is a candidate paracrine regulator(9) , expression of the activin betaA subunit increases in parenchymal hepatocytes; therefore, activin appears to be a candidate for an autocrine regulator of hepatocyte proliferation(10) . The active receptor complex for homodimeric ligands of the TGF-beta family including activin appears to obligatorily involve a heterodimeric complex of two genetically distinct kinases which have been designated type I and type II according to sequence homology and size(4, 5, 6, 7, 8) . Our laboratory cloned two type I serine/threonine kinase receptors, SKR1 (12) and SKR2(13) , from a well differentiated human hepatoma cell, HepG2, which exhibits many properties of parenchymal hepatocytes. We have shown that four SKR2 variants, SKR2-1, SKR2-2, SKR2-3, and SKR2-4, arise by alternative splicing and poly(A) addition and vary in carboxyl-terminal domains, potential phosphorylation sites, and kinase activities(13) . Subsequent to our work, SKR1 was also cloned from different species and named as R1(14) , TsK7L(15) , ALK2(16) , or ActXIR(17) . The homolog of SKR2-1 has been designated as R2(14) , ActR-IB(18) , or ALK4 (16, 19) by others. Although inactive alone, SKR1 and SKR2 were subsequently shown to bind TGF-beta1 or activin in concert with the TGF-beta1 or activin type II receptor, respectively(6, 7, 8) . (^2)However, in transfected mammalian cell types examined so far, both SKR1 and SKR2 appear to elicit biological effects only in concert with the activin type II receptor (ActRII) and activin(7, 18, 19) .

Artificially constructed monomers with defects in catalytic activity or substrate binding that heterodimerize with a native subunit have been widely employed as dominant-negative inhibitors to demonstrate the requirement for dimeric transmembrane receptors in signal transduction. A dominant-negative effect of an artificial mutant subunit of the homodimeric ligand, platelet-derived growth factor, has been reported (20) . However, reports of naturally occurring dominant-negative mechanisms of regulation of ligand-activated transmembrane receptors are rare(21, 22) . We report here that the inhibin heterodimer has properties of a natural dominant-negative inhibitor of the heterodimeric type II-type I receptor kinase complex promoted by homodimeric activin in liver cells.


EXPERIMENTAL PROCEDURES

Overexpression of Recombinant ActRII and Ectodomain of SKR2 in Baculoviral-infected Insect Cells

The full-length coding sequence of mouse ActRII cDNA (4) was subcloned into the NotI/BamHI sites of baculoviral transfer plasmid pVL-1392 (Invitrogen). A human SKR2 cDNA was amplified from the cloned SKR2 cDNA template (13) in the polymerase chain reaction using 5` (5`-TATGAATTCGGTTACTATGGCGGAGTCGGC-3`) and 3` primers (5`-TAAGGATCCCTCTTGTAAAACGATGGTTCG-3`). The resultant cDNA spanned the sequence from 7 base pairs upstream of the translational initiation site through coding sequence for 62 residues of the intracellular juxtamembrane domain. To facilitate immunochemical analysis, a portion of human fibroblast growth factor receptor cDNA coding for the M1C4 monoclonal antibody epitope (23) was ligated in-frame with the SKR2 cDNA at its 3`-end at a BamHI site. The tagged SKR2 cDNA was cloned into the EcoRI sites of pVL-1392. Recombinant baculoviruses bearing ActRII and SKR2 cDNAs were prepared as described elsewhere(23) . Except where indicated, the infected Sf9 cells were harvested within 65 h. The epitope-tagged chimeric protein containing SKR2 extracellular, transmembrane, and intracellular juxtamembrane domains from cell lysates was analyzed by immunoblot and immunoprecipitation methods by using monoclonal antibody M1C4 as described previously(23) .

Ligand Binding and Covalent Affinity Cross-linking

Recombinant human activin A and inhibin A were radiolabeled with I-iodine as described elsewhere(12) . Unless otherwise indicated, baculoviral infected Sf9 cells (7.5 times 10^5) were suspended in 0.5 ml of binding buffer (50 mM HEPES (pH 7.5), 128 mM NaCl, 5 mM KCl, 5 mM MgSO(4), 1.2 mM CaCl(2), 2 mg/ml bovine serum albumin) and mixed with 0.5 pmol of I-labeled activin (2.38 times 10^6 cpm/pmol) or inhibin (3.84 times 10^6 cpm/pmol) for 90 min at room temperature in the absence or presence of an excess amount of unlabeled ligand. The bound ligand was covalently cross-linked to receptor sites with 0.5 mM disuccinimidyl suberate in binding buffer without bovine serum albumin. The cells were extracted with ice-cold cell lysis buffer (10 mM Tris-HCl (pH 7.0), 125 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 µg/ml leupeptin, 50 µg/ml aprotinin, 0.3 mM phenylmethylsulfonyl fluoride). The affinity-labeled complexes from 7.5 times 10^5 cells were analyzed by immunoprecipitation, SDS-PAGE, and autoradiography(23) . Monolayers of HepG2 cells were affinity-labeled with I-labeled activin or inhibin by using the same procedure.

Immunoprecipitation

Lysates of Sf9 cells (3 times 10^6) from binding assays were absorbed with normal rabbit IgG immobilized to protein A-Sepharose CL-4B beads (Pharmacia Biotech Inc.) before reaction with the M1C4 monoclonal antibody immobilized to protein A-Sepharose as previously described(23) . Lysates (0.25 ml) of HepG2 cells (2 times 10^6) prepared as described above were adjusted to 0.3% SDS, boiled for 4 min, diluted 2-fold, and clarified by centrifugation at 16,000 times g for 5 min at 4 °C. The supernatant was then mixed with 35 µl of protein A beads and 10 µl of anti-SKR2 serum N19S or serum from the same rabbit obtained prior to immunization. Antiserum was prepared against a synthetic polypeptide sequence N19S (NRIDLRVPSGHLKEPEHPS) in the extracellular juxtamembrane of SKR2 (13) by previously described methods(23) . After incubation for 3 h at 4 °C, the beads were extracted and analyzed by SDS-PAGE and autoradiography(23) .

Untreated and SDS- and heat-treated lysates of HepG2 cells described above were also subjected to analysis by immunoprecipitation with anti-ActRII monoclonal antibody, mAb149/1, as described elsewhere(24) . The conditioned medium of hybridoma mAb149/1 was a generous gift from Dr. R. Bicknell (Imperial Cancer Research Fund, Oxford, UK). For immunoprecipitations, 0.30 ml of cell lysate was mixed with 0.15 ml of the mAb149/1-conditioned medium and 35 µl of goat anti-mouse IgG-agarose beads (Sigma).

DNA Synthesis

Acid-insoluble [^3H]thymidine incorporation was determined in 1.5 times 10^4 HepG2 cells in 24-well plates containing 1 ml of minimum Eagle's medium and 1% fetal bovine serum after 3 days of exposure to activin, inhibin, or both at the indicated concentrations. Cells were incubated at 37 °C for 4 h with 0.5 µCi added to each well in 10 µl of phosphate-buffered saline. After a wash with 1 ml of phosphate-buffered saline, cells were soaked in 1 ml of 10% trichloroacetic acid at 4 °C for 30 min, then solubilized in 0.25 ml of 0.5 N NaOH, and the solution was counted by liquid scintillation.


RESULTS AND DISCUSSION

Activin A Homodimer Binds to Recombinant ActRII and the Ectodomain of SKR2

To study the binding of activin and inhibin to ActRII and SKR2, full-length recombinant ActRII protein and an epitope-tagged SKR2 chimeric protein exhibiting the extracellular, transmembrane, and intracellular juxtamembrane domains of SKR2 and a specific monoclonal antibody epitope M1C4 at its COOH terminus (23) were expressed in baculoviral infected Sf9 insect cells. Western blot analyses with ActRII-specific monoclonal antibody mAb149/1 (24) and SKR2 chimeric protein-specific monoclonal antibody M1C4 (23) indicated that the apparent molecular masses of the insect cell-expressed recombinant ActRII and the tagged SKR2 ectodomain on SDS-PAGE were 72 and 43 kDa, respectively. Table 1lists the possible complexes that can arise and their relative abundance observed experimentally from interaction of activin A, inhibin A, ActRII, and SKR2 after incubation and subsequent exposure to a covalent affinity cross-linking reagent followed by analysis of stable complexes by SDS-PAGE and autoradiography. When expressed alone, the recombinant ectodomain of SKR2 failed to bind I-labeled activin A (Fig. 1A). Immunoassay with the M1C4 antibody confirmed the presence of the 43-kDa antigen in the infected cell lysates (results not shown). Cells expressing the 72-kDa ActRII product exhibited I-labeled bands of 98 and 85 kDa which correlated with the size of the ActRII product cross-linked to a 28-kDa beta:beta homodimer and a single 14-kDa beta chain of activin, respectively (Fig. 1A). The results suggest that beta:beta activin binds to ActRII through one of its subunits. The significant band at 28 kDa above the dye front containing the 14-kDa beta chain monomer of activin indicated that the two disulfide-linked beta chains of the homodimer can be cross-linked when bound to ActRII or in free nonspecifically bound form (Fig. 1A). Significant amounts of higher molecular mass ligand-labeled material were apparent at the top of the gels; however, these analyses could neither confirm the presence of the RIIbulletbeta:betabulletRII species (Table 1) nor shed light on the stoichiometry of the binding of ActRII to each beta chain within the activin homodimer. Cells which were co-infected with both viruses bearing epitope-tagged chimeric SKR2 and ActRII cDNAs exhibited an additional band at 57 kDa which is the size predicted for the 43-kDa epitope-tagged SKR2 product linked to one 14-kDa beta chain (Fig. 1A). A 70-kDa band indicative of SKR2 linked to a 28-kDa beta:beta activin homodimer was notably absent. Immunoprecipitation with monoclonal antibody M1C4 confirmed that the 57-kDa band contained the SKR2 product (Fig. 1B). In addition, the 85-kDa complex of an I-labeled activin beta chain and ActRII coprecipitated with the anti-SKR2 M1C4 antibody (Fig. 1B). In contrast to cell lysates, the 98-kDa band of ActRII bearing the activin beta:beta dimer was barely detectable in the immunoprecipitates. In contrast to the lysates, the relative intensity of the 57- and 85-kDa bands in the immunoprecipitates was near 1:1 at all three ratios of co-infection. The results confirmed that the binding of beta:beta activin to ActRII facilitates binding of SKR2 to the ActRII-activin complex in stoichiometric amounts. Although we cannot eliminate the possibility that an ActRII and SKR2 complex is bound to each beta chain of the activin beta:beta dimer, the absence of a significant band at 128-kDa indicative of a ActRIIbulletbetabulletSKR2 complex (Fig. 1B and results not shown) suggests an ActRIIbulletbeta:betabulletSKR2 complex in which ActRII and SKR2 are bound respectively to each chain of the beta:beta dimer. The absence of the SKR2bulletbeta:beta species in both lysates and immunoprecipitates and the ActRIIbulletbeta:beta species in the immunoprecipitates may indicate a spatial relationship between the two beta chains of activin when bound to both ActRII and SKR2 that reduces the efficiency of interchain cross-linking.




Figure 1: Binding of I-labeled activin to recombinant ActRII and ectodomain of SKR2 in baculoviral-infected Sf9 insect cells. A, radiolabeled bands from cell lysates. B, radiolabeled bands precipitated with an SKR2 antibody. Cells were infected with virus-bearing ActRII and SKR2 cDNA as indicated. Antisense indicates cells infected with virus bearing SKR2 cDNA in the antisense orientation. Different infection ratios of ActRII and SKR2 viral titers at a constant titer of ActRII virus are indicated at the bottom. Where indicated, unlabeled activin A was present in 100-fold excess of the I-labeled activin. Affinity-labeled complexes and immunoprecipitation samples were analyzed by SDS-PAGE and autoradiography. Molecular mass standards are indicated in kilodaltons.



Inhibin A alpha:beta Heterodimer Binds to ActRII but Not SKR2

I-Labeled inhibin covalently cross-linked to bands of 105 and 85 kDa on Sf9 cells which were infected with virus bearing the ActRII cDNA (Fig. 2A). The major 105-kDa band was the predicted size of 72-kDa ActRII bearing the 32-kDa alpha:beta inhibin heterodimer while the 85-kDa band correlated with that of ActRII cross-linked to the 14-kDa beta subunit of inhibin (Table 1). No distinct 90-kDa band indicative of ActRII linked to the unique monomeric 18-kDa alpha chain of inhibin could be detected. In addition, high molecular material at the top of the gels indicative of oligomers of ActRII and the labeled alpha:beta inhibin ligand was much less than the same analyses employing activin as ligand (Fig. 1A). The intense band at 32 kDa as well as the high yield of the ActRIIbulletbeta:alpha complex (105 kDa) indicated that, similar to activin, disuccinimidyl suberate efficiently cross-linked the alpha and beta subunits of inhibin to each other when bound both to ActRII or nonspecifically as the free ligand (Fig. 2A, Table 1). The high intensity of the ActRIIbulletbeta:alpha species relative to the ActRIIbulletbeta complex probably indicates a higher specific activity of the I-labeled alpha chain relative to the beta chain. Neither cells infected with single virus bearing the SKR2 cDNA nor those co-infected with viruses bearing SKR2 and ActRII cDNAs yielded SKR2 bands labeled with inhibin in the total cell lysates (Fig. 2A) or in immunoprecipitates with the M1C4 antibody (Fig. 2B). The results suggest that inhibin binds to ActRII through its beta subunit which is shared with activin, but inhibin cannot promote formation of the ActRIIbulletSKR2 heterodimer because of inability of SKR2 to bind to the inhibin alpha chain. Although steric interference by presence of the unique alpha chain cannot be eliminated, these results further suggest that it is unlikely that ActRII and SKR2 assemble on a single beta chain of the activin beta:beta homodimer.


Figure 2: Binding of I-labeled inhibin to recombinant ActRII and ectodomain of SKR2. A, radiolabeled bands from cell lysates. B, radiolabeled bands precipitated with M1C4 antibody. Conditions were the same as in Fig. 1except that I-labeled and unlabeled inhibin A was substituted for activin A.



Activin A and Inhibin A Compete for Binding to Recombinant ActRII

High levels of nonspecific binding of labeled activin and inhibin impaired a direct comparative Scatchard analysis of the affinity of the two ligands for ActRII. As an alternative, the ability of unlabeled activin and inhibin to reduce the amount of labeled activin that was covalently cross-linked to ActRII was performed. Both activin-labeled ActRII bands at 98 and 85 kDa in baculoviral infected Sf9 cells (Fig. 1A and Fig. 3, lane 2) were reduced by an excess of unlabeled activin (Fig. 3, lanes 3-6) and inhibin (Fig. 3, lanes 7-10). In experiments not shown here, the same results were achieved when Sf9 cells were co-infected with baculoviruses bearing ActRII and SKR2 cDNAs. Activin-labeled ActRII and SKR2 bands were proportionally reduced by unlabeled activin or inhibin. Quantitative analysis of the results by scanning densitometry revealed that on a molar basis 2-4 times more inhibin than activin was required to reduce the activin-labeled ActRII complexes to the same level (Fig. 3). Currently, it is unclear whether this reflects an intrinsically lower affinity of the inhibin beta chain for ActRII due to influence of the alpha chain or that the molar ratio of binding of ActRII to the beta:beta activin homodimer is greater than one.


Figure 3: Competition of inhibin and activin for binding of activin to recombinant ActRII. Sf9 cells (5 times 10^5) were infected with baculoviruses bearing antisense (lane 1) or ActRII (lanes 2-10) cDNAs and incubated for 90 min at room temperature in binding buffer containing 0.30 pmol of I-labeled human recombinant activin and the indicated amounts in picomoles of unlabeled activin (lanes 3-6) or inhibin (lanes 7-10) in a total volume of 0.25 ml. After addition of DSS, the I-activin-labeled complexes were analyzed by SDS-PAGE and autoradiography. The relative intensities of the radiolabeled 98- and 85-kDa ActRII bands (bottom) was determined by scanning densitometry across the area of the film containing the two ActRII bands.



Activin and Inhibin Receptors in HepG2 Cells

To test whether native activin and inhibin receptors in liver cells exhibited properties similar to recombinant ActRII and SKR2, analyses were carried out in human hepatoma cells (HepG2) which express ActRII and from where SKR2 was originally cloned(12, 13) . Both labeled activin and inhibin yielded a labeled 89-kDa band, activin labeled specific bands at 72 and 165 kDa, and inhibin labeled a specific band at 110 kDa. All four bands were eliminated by addition of unlabeled activin or inhibin to the binding reaction (Fig. 4A). The common 89-kDa band labeled by both ligands had the predicted molecular mass of a 75-kDa mammalian ActRII linked to the common 14-kDa beta subunit of activin and inhibin. The 72-kDa band that was specifically labeled with I-labeled activin was characteristic of a 59-kDa type I activin receptor linked to a single 14-kDa beta chain. Since the 165-kDa weak band that was uniquely labeled with activin is sensitive to unlabeled inhibin and near the sum of the 89- and 72-kDa bands, it is likely the heterodimer of type II and type I activin receptors bearing the homodimeric beta:beta activin ligand. The 110-kDa band that labeled uniquely with I-labeled inhibin correlates with the size of a complex of the 75-kDa ActRII monomer and the 32-kDa alpha:beta inhibin heterodimer. All three activin-labeled bands precipitated with an antiserum (N19S) against the extracellular juxtamembrane of SKR2 (Fig. 4B). No inhibin-labeled bands were precipitated with the antiserum. In contrast to results in Fig. 1B and Fig. 2B using the M1C4 monoclonal antibody, efficient reactivity of ligand-labeled complexes with the N19S anti-serum required denaturation of extracts prior to immunoprecipitation. This resulted in loss during the immunoprecipitation of the fraction of the cross-linked 89-kDa ActRIIbulletbeta species from ActRIIbulletbeta:betabulletSKR2 complexes in which the adjacent beta chain was not concurrently cross-linked to SKR2 (Fig. 4B). This was reflected in a lower yield of the 89-kDa ActRIIbulletbeta complex relative to the SKR2bulletbeta band at 72 kDa. The low yield of the 165-kDa ActRIIbulletbeta:betabulletSKR2 complexes that survive the immunoprecipitation and SDS-PAGE analysis may reflect (a) the lower probability of concurrent covalent cross-linking events occurring between ActRIIbulletbeta, SKR2bulletbeta, and the two beta chains of homodimeric activin within the same complex or (b) a low efficiency of inter-beta chain cross-linking within the beta:beta homodimer when it is bound to both ActRII and SKR2 (discussed earlier).


Figure 4: Characterization of activin and inhibin receptors in human liver cells (HepG2). A, radiolabeled receptor sites from cell lysates. Monolayers of HepG2 cells (1 times 10^6) were incubated with I-labeled activin or inhibin in the absence or presence of a 100-fold excess of unlabeled ligand as indicated and radiolabeled binding sites were analyzed as described in Fig. 1. B, immunoprecipitation of ligand-labeled sites with SKR2 antiserum. The denatured lysates of HepG2 cells (2 times 10^6) from binding assays were prepared and subjected to immunoprecipitation by using SKR2-specific antiserum N19S or preimmune serum from the same rabbit (negative control). C, immunoprecipitation of ligand-labeled binding sites with anti-ActRII monoclonal antibody. The anti-ActRII mAb149/1 was used to precipitate both I-activin and inhibin-labeled bands from HepG2 cell lysates. An equal amount of normal mouse IgG was used as negative control. The dried gel was exposed to a storage phosphor screen and visualized by screening on a Molecular Dynamics PhosphorImager.



In contrast to the anti-SKR2 antibody (N19S), a monoclonal antibody against ActRII (mAb149/1) precipitated the three activin-labeled bands at 72, 89, and 165 kDa and the inhibin-labeled bands at 89 and 110 kDa from the HepG2 cell lysates (Fig. 4C). In addition, the anti-ActRII antibody revealed a weak activin-labeled band at 101 kDa that varied in intensity among assays (Fig. 4C). The 101-kDa band is characteristic of a complex of ActRII and the beta:beta activin dimer that was apparent in whole lysates of the baculoviral infected Sf9 cells (Fig. 1). mAb149/1 precipitated labeled ActRII (89 kDa) and activin type I receptor (ActRI) complexes (72 kDa) in a 1:1 ratio from undenatured cell lysates, but yielded predominately the labeled ActRIIbulletbeta complex from lysates that were denatured prior to immunoprecipitation for reasons discussed above in the analysis with the anti-SKR2 antibody N19S (Fig. 4C).

I-Activin-labeled 72-kDa (type I), 89-kDa (type II), and 165-kDa (types I and II complex) bands from the HepG2 cells were proportionally reduced by both unlabeled activin and inhibin in a dose-dependent manner (Fig. 5). Similar to the results with recombinant ActRII and SKR2 expressed in the Sf9 insect cells, quantitation of relative band intensities indicated that inhibin was 3-4 times less effective than activin in the ability to compete with labeled activin for binding to ActRII (Fig. 5).


Figure 5: Competition of the binding of activin to HepG2 cells with inhibin. Monolayers of HepG2 cells (1 times 10^6) in 60-mm dishes were incubated with 0.4 pmol of I-labeled activin in the presence of the indicated amounts in picomoles of unlabeled activin (lanes 2-5) or inhibin (lanes 6-9) in a total volume of 0.20 ml for 90 min at room temperature. Affinity-labeled complexes from cell lysates were analyzed by a PhosphorImager as described in Fig. 4. Relative intensities (below) of the 89/72-kDa receptor bands at each level of competitor was determined from the image by densitometry.



These results confirm that activin and inhibin share the same native type II receptor in liver cells; inhibin blocks the binding of activin to type II receptors and thereby prevents heterodimerization of the type II and type I receptors. HepG2 cells exhibit neither a ligand-specific type II receptor nor a type I receptor for inhibin that can be detected by covalent affinity cross-linking.

Antagonistic Effects of Activin and Inhibin on HepG2 Cell Growth

Activin inhibited DNA synthesis in the HepG2 cells in a dose-dependent fashion (Fig. 6A). Inhibition was half-maximal at about 100 pM activin and reached a maximum at 300 pM (Fig. 6A). In contrast, inhibin had no effect on DNA synthesis at concentrations up to 2.5 nM (Fig. 6B). However, at 300 pM activin in which DNA synthesis was maximally inhibited, inhibin antagonized the effect of activin (Fig. 6B). About a 2 times molar ratio of inhibin to activin was required to antagonize the inhibitory effect of activin by 50% (Fig. 6B). This result is consistent with the ability of inhibin to compete with activin for binding to ActRII and inhibit formation of the ActRII.beta:betabulletActRI receptor complex in both baculoviral infected insect cells and the HepG2 cells.


Figure 6: Antagonistic effect of inhibin on inhibition of HepG2 cell DNA synthesis by activin. A, HepG2 cells (1.5 times 10^4 each) were treated with activin at the indicated concentrations of 37.5, 75, 150, 300, and 600 pM for 3 days and [^3H]thymidine incorporation was determined as described under ``Experimental Procedures.'' Data points are the mean of duplicates and presented as a percent of DNA synthesis in controls containing no activin (100%). B, inhibin at 150, 300, 600, 1200, and 2400 pM was added to HepG2 cell cultures in the absence of activin (circles) or in the presence of 300 pM activin (triangles) for 3 days, and then the incorporation of [^3H]thymidine was determined.



Conclusions

We suggest that some, if not all, biological activities of inhibin may be mediated by its role as an inhibitor of formation of the active heterodimeric activin receptor complex of type II and type I serine/threonine kinases (Fig. 7). We have shown that SKR2, a widely expressed activin type I serine/threonine kinase(13, 18) , is recruited into an ActRIIbulletbeta:betabulletSKR2 complex by association with a beta chain of the activin beta:beta homodimer (Fig. 7). Recently, SKR2 (also called ActRIB) in conjunction with ActRII has been shown to be a mediator of the growth-inhibitory and extracellular matrix transcriptional responses of cells to activin(18, 25) . Through its beta chain subunit which is shared with activin, the inhibin alpha:beta heterodimer competes with activin for binding to activin type II receptors, inhibits formation of the ActRIIbulletbeta:betabulletActRI complex and thereby inhibits the activin-dependent response.


Figure 7: Hypothetical scheme of inhibition of assembly of the heterodimeric activin receptor complex by inhibin. Activin beta:beta homodimer binds to the type II receptor through one of the beta subunits which induces capacity of the unoccupied beta subunit to bind the type I receptor (SKR2 in the current study). The alpha subunit of inhibin is incapable of binding type I receptors and results in a dominant-negative inhibition of assembly of the heterodimeric activin receptor.



In addition to significant differences in size and sequence, the unique alpha chain of inhibin differs from the common beta chain of activin and inhibin by absence of the two half-cystines which form the disulfide which anchors the NH(2)-terminal alpha1 helix to the rest of the monomer structure(26) . Homodimeric ligands, TGF-beta1 and TGF-beta2, which also bind to SKR2 when anchored to the TGF-beta type II receptor,^2 exhibit a similar structure in this domain. The conformation of the NH(2)-terminal domain may be important in the binding of one chain within the homodimeric ligands within the TGF-beta family to type I receptors, while the other chain is anchored to the appropriate type II receptor. The domain may be a target for alteration and design of mutant:wild-type heterodimeric antagonists of TGF-beta ligands.


FOOTNOTES

*
This work was supported by United States Public Health Service NIDDK grants DK35310 and DK38639 and by National Cancer Institute Grant CA59971. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

To whom correspondence should be addressed: Albert B. Alkek Institute of Biosciences and Technology, Texas A& University, 2121 W. Holcombe Blvd., Houston, TX 77030-3303. Tel.: 713-677-7522; Fax: 713-677-7512; wmckeeha{at}ibt.tamu.edu.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; SKR, serine/threonine kinase receptor; ActRII, activin receptor type II; ActRI, activin receptor type I; cDNA, complementary DNA; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. Xu, K. Matsuzaki, and W. L. McKeehan, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. W. Vale (Salk Institute, San Diego, CA) for the mouse ActRII cDNA, Dr. R. Bicknell (Imperial Cancer Research Fund, Oxford, UK) for mAb149/1, and Drs. L. Krummen and J. Mather (Genentech, South San Francisco, CA) for recombinant activin A and inhibin A.


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