Reconstitution and analysis of soluble inhibin and activin receptor complexes in a cell-free system

Activins and inhibins compose a heterogeneous subfamily within the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors with critical biological activities in embryos and adults. They signal through a heteromeric complex of type II, type I, and for inhibin, type III receptors. To characterize the affinity, specificity, and activity of these receptors (alone and in combination) for the inhibin/activin subfamily, we developed a cell-free assay system using soluble receptor-Fc fusion proteins. The soluble activin type II receptor (sActRII)-Fc fusion protein had a 7-fold higher affinity for activin A compared with sActRIIB-Fc, whereas both receptors had a marked preference for activin A over activin B. Although inhibin A and B binding was 20-fold lower compared with activin binding to either type II receptor alone, the mixture of either type II receptor with soluble TGF-beta type III receptor (TbetaRIII; betaglycan)-Fc reconstituted a soluble high affinity inhibin receptor. In contrast, mixing either soluble activin type II receptor with soluble activin type I receptors did not substantially enhance activin binding. Our results support a cooperative model of binding for the inhibin receptor (ActRII.sTbetaRIII complex) but not for activin receptors (type II + type I) and demonstrate that a complex composed of activin type II receptors and TbetaRIII is both necessary and sufficient for high affinity inhibin binding. This study also illustrates the utility of this cell-free system for investigating hypotheses of receptor complex mechanisms resulting from crystal structure analyses.     

In the absence of type III receptor, the transforming growth factor (TGF)-beta type II-B receptor requires the type I receptor to bind TGF-beta2

Transforming growth factor beta (TGF-beta) ligands exert their biological effects through type II (TbetaRII) and type I receptors (TbetaRI). Unlike TGF-beta1 and -beta3, TGF-beta2 appears to require the co-receptor betaglycan (type III receptor, TbetaRIII) for high affinity binding and signaling. Recently, the TbetaRIII null mouse was generated and revealed significant non-overlapping phenotypes with the TGF-beta2 null mouse, implying the existence of TbetaRIII independent mechanisms for TGF-beta2 signaling. Because a variant of the type II receptor, the type II-B receptor (TbetaRII-B), has been suggested to mediate TGF-beta2 signaling in the absence of TbetaRIII, we directly tested the ability of TbetaRII-B to bind TGF-beta2. Here we show that the soluble extracellular domain of the type II-B receptor (sTbetaRII-B.Fc) bound TGF-beta1 and TGF-beta3 with high affinity (K(d) values = 31.7 +/- 22.8 and 74.6 +/- 15.8 pm, respectively), but TGF-beta2 binding was undetectable at corresponding doses. Similar results were obtained for the soluble type II receptor (sTbetaRII.Fc). However, sTbetaRII.Fc or sTbetaRII-B.Fc in combination with soluble type I receptor (sTbetaRI.Fc) formed a high affinity complex that bound TGF-beta2, and this complex inhibited TGF-beta2 in a biological inhibition assay. These results show that TGF-beta2 has the potential to signal in the absence of TbetaRIII when sufficient TGF-beta2, TbetaRI, and TbetaRII or TbetaRII-B are present. Our data also support a cooperative model for receptor-ligand interactions, as has been suggested by crystallization studies of TGF-beta receptors and ligands. Our cell-free binding assay system will allow for testing of models of receptor-ligand complexes prior to actual solution of crystal structures.     

Regulation of TGF-beta ligand and receptor expression in neonatal rat lungs exposed to chronic hypoxia.

Long-term effects of hypoxia are largely due to its modulatory effects on proliferation and differentiation of epithelial and endothelial cells, processes also regulated by the transforming growth factor (TGF)-beta system. We investigated the effects of hypoxia on the TGF-beta system in rat lungs from different developmental stages. Sprague-Dawley rats were exposed to 9.5% oxygen during either the first 2 wk of life or adulthood. Analysis revealed an arrest of alveolarization in hypoxic postnatal day 14 rats. Bioactive TGF-beta levels in bronchoalveolar lavage fluid were increased in these animals, and Western blot analysis revealed upregulation of TGF-beta receptor (TbetaR) I and II. None of these changes was observed in hypoxic adults. Hypoxia did, however, lead to decreased expression of TbetaRIII in both postnatal day 14 and adult rats. Immunohistochemical analysis localized TbetaRI-III predominantly to bronchiolar and alveolar epithelium; these patterns did not change with hypoxia. Thus we observed changes in TGF-beta activity and TbetaR isotype expression in rat lung that parallel the arrest in alveolarization seen with chronic hypoxia in early development. These alterations may partly explain the morphological changes observed in hypoxia.     

Bone morphogenetic proteins signal through the transforming growth factor-beta type III receptor

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-beta (TGF-beta) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-beta type III receptor (TbetaRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-beta and inhibin receptor. Here we demonstrate that TbetaRIII functions as a BMP cell surface receptor. TbetaRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-beta. TbetaRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TbetaRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TbetaRIII function, we demonstrate that BMP-2 specifically stimulates TbetaRIII-mediated epithelial to mesenchymal cell transformation. The ability of TbetaRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-beta signaling suggests a broader role for TbetaRIII in orchestrating TGF-beta superfamily signaling.     

Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form

Transforming growth factor-beta (TGF-beta) is a key modulator of epidermal development and homeostasis, and has been shown to potently regulate keratinocyte migration and function during wound repair. There are three cloned TGF-beta receptors termed type I, type II, and type III that are found on most cell types. The types I and II are the signaling receptors, while the type III is believed to facilitate TGF-beta binding to the types I and II receptors. Recently, we reported that in addition to these receptors, human keratinocytes express a 150 kDa TGF-beta 1 binding protein (r150) which forms a heteromeric complex with the TGF-beta signaling receptors. This accessory receptor was described as glycosyl phosphatidylinositol-specific anchored based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). In the present study, we demonstrate that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta 1 with an affinity similar to those of the types I and II TGF-beta signaling receptors. Furthermore, the PIPLC released (soluble) form of this protein is capable of binding TGF-beta 1 independently from the signaling receptors. In addition, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Our observation that r150 interacts with the TGF-beta signaling receptors, together with the finding that the soluble r150 binds TGF-beta 1 suggest that r150 in either its membrane anchored or soluble form may potentiate or antagonize TGF-beta signaling. Elucidating the mechanism by which r150 functions as an accessory molecule in TGF-beta signaling may be critical to understanding the molecular mechanisms underlying the regulation of TGF-beta action in keratinocytes.     

Two novel patterns of transforming growth factor beta (TGF-beta) binding to cell surface proteins are dependent upon the binding of TGF-beta 1 and indicate a mechanism of positive cooperativity.

Three isoforms of the transforming growth factor beta (TGF-beta) family, TGF-beta 1, TGF-beta 2, and TGF-beta 3, bind specifically and with high affinity to several cell surface components known as type I, type II, and type III proteins. The type I and II proteins may serve as biological receptors, whereas the type III protein does not appear to be associated with TGF-beta-mediated cell responses, and its function remains unknown. Binding data on confluent monolayers of rat skeletal myoblasts of the L6 cell line reveals two novel patterns of TGF-beta 1 binding. Saturation of the type I receptor with native TGF-beta 2 induces a 7-fold increase in binding of radiolabeled TGF-beta 1 at the type II protein. No induction of type II receptor binding was observed on subconfluent cells indicating a density-dependent phenomenon. The data suggest that the type I and type II proteins may interact during ligand binding in a manner which may be indicative of a regulatory role that is activated by the phase of cell growth or differentiation. A second observation is the binding of TGF-beta to a glycoprotein of 180 kDa and referred to here as the "type VI" binding protein. This protein is not related to previously described TGF-beta binding proteins, and its distribution appears universal among cell types. The level of TGF-beta 1 binding to this protein is dependent on the presence of TGF-beta 2. It is not known whether this protein transmits biological information or whether it serves as an accessory protein of a TGF-beta receptor complex.     

Restoration of expression of transforming growth factor-beta type II receptor in murine renal cell carcinoma (renca) cells by 5-Aza-2'-deoxycytidine

The murine renal cell carcinoma (Renca) cells are insensitive to TGF-beta due to a lack of TGF-beta type II receptor (TbetaR-II). The objective of the present study is to determine the mechanism of this loss of sensitivity to TGF-beta in Renca cells. Renca cells were cultured and treated with 5-Aza-2'-Deoxycytidine (5-Aza), a specific inhibitor of methylation. Expression of TGF-beta type I receptor (TbetaRI) and TbetaRII was determined by RT-PCR and Western blot analysis before and after the treatment of Renca cells with 5-Aza. The expression of phosphorylated Smad2 (P-Smad2) was determined by Western blot analysis. TGF-beta levels in the conditioned medium were measured by ELISA. Renca cells did not express TbetaR-II prior to 5-Aza treatment. After 5-Aza treatment, these cells expressed TbetaR-II at both mRNA and protein levels, which corresponded to the restoration of sensitivity to TGF-beta by an increase in P-Smad2. Levels of TGF-beta1 were similar before and after 5-Aza treatment. Results of the present study indicated that, in Renca cells, the loss of sensitivity to TGF-beta is likely due to a promoter hypermethylation in the TbetaR-II gene.     

Hormone induced changes in lactase glycosylation in developing rat intestine

Lactase exists in both soluble and membrane-bound forms in suckling rat intestine. The distribution of lactase and its glycosylated isoforms in response to thyroxine or cortisone administration has been studied in suckling rats. 75% of lactase activity was detected, associated with brush borders, compared to 24% in the soluble fraction of 8-day-old rats. Thyroxine treatment enhanced soluble lactase activity to 34%, whereas particulate fraction was reduced to 67% compared to controls. Cortisone administration reduced soluble lactase activity from 24% in controls to 12% with a concomitant increase in membrane-bound activity to 89%. Western blot analysis revealed lactase signal, corresponding to 220 kDa in both the soluble and membrane fractions, which corroborated the enzyme activity data. The elution pattern of papain solubilized lactase from agarose-Wheat Germ agglutinin, or Concanavalin A or Jacalin agglutinin columns was different in the suckling and adult rat intestines. Also the elution profile of lactase activity from agarose-lectin columns was modulated in cortisone, thyroxine, and insulin injected pups, which suggests differences in glycosylated isoforms of lactase under these conditions. These findings suggest the role of these hormones in inducing changes in lactase glycosylation during postnatal development of intestine, which may contribute to adult-type hypolactasia in rats.     

Syndecan-2 regulates transforming growth factor-beta signaling

Transforming growth factor-beta (TGF-beta) has multiple functions including increasing extracellular matrix deposition in fibrosis. It functions through a complex family of cell surface receptors that mediate downstream signaling. We report here that a transmembrane heparan sulfate proteoglycan, syndecan-2 (S2), can regulate TGF-beta signaling. S2 protein increased in the renal interstitium in diabetes and regulated TGF-beta-mediated increased matrix deposition in vitro. Transfection of renal papillary fibroblasts with S2 or a S2 construct that has a truncated cytoplasmic domain (S2DeltaS) promoted TGF-beta binding and S2 core protein ectodomain directly bound TGF-beta. Transfection with S2 increased the amounts of type I and type II TGF-beta receptors (TbetaRI and TbetaRII), whereas S2DeltaS was much less effective. In contrast, S2DeltaS dramatically increased the level of type III TGF-beta receptor (TbetaRIII), betaglycan, whereas S2 resulted in a decrease. Syndecan-2 specifically co-immunoprecipitated with betaglycan but not with TbetaRI or TbetaRII. This is a novel mechanism of control of TGF-beta action that may be important in fibrosis.     

Oncogenic Ki-ras confers a more aggressive colon cancer phenotype through modification of transforming growth factor-beta receptor III

Transforming growth factor-beta1 (TGF-beta1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. Each of three Ki-ras(G12V) transfectants of HD6-4 colon cancer cells had been shown to be more aggressive in vivo than controls in earlier studies (Yan, Z., Chen, M., Perucho, M., and Friedman, E. (1997) J. Biol. Chem. 272, 30928-30936). We now show that stable expression of oncogenic Ki-ras(G12V) converts the HD6-4 colon cancer cell line from insensitive to TGF-beta1 to growth-promoted by TGF-beta1. Each of three Ki-ras(G12V) transfectants responded to TGF-beta1 by an increase in proliferation and by decreasing the abundance of the Cdk inhibitor p21 and the tumor suppressor PTEN, whereas each of three wild-type Ki-ras transfectants remained unresponsive to TGF-beta1. The wild-type Ki-ras transfectants lack functional TGF-beta receptors, whereas all three Ki-ras(G12V) transfectants expressed functional TGF-beta receptors that bound (125)I-TGF-beta1. The previous studies showed that in cells with wild-type Ki-ras, TGF-beta receptors were not mutated, and receptor proteins were transported to the cell surface, but post-translational modification of TGF-beta receptor III (TbetaRIII) was incomplete. We now show that the betaglycan form of TbetaRIII is highly modified following translation when transiently expressed in Ki-ras(G12V) cells, whereas no such post-translational modification of TbetaRIII occurs in control cells. Antisense oligonucleotides directed to Ki-Ras decreased both TbetaRIII post-translational modification in Ki-ras(G12V) cells and TGF-beta1 down-regulation of p21, demonstrating the direct effect of mutant Ras. Therefore, one mechanism by which mutant Ki-Ras confers a more aggressive tumor phenotype is by enhancing TbetaRIII post-translational modification.     

Ectodomain cleavage and shedding of the type III transforming growth factor-beta receptor in lung membranes effect of temperature, ligand binding and membrane solubilization.

Previous studies from our laboratory [Philip, A. & O'Connor-McCourt, M. D. (1991) J. Biol. Chem. 266, 22290--22296] have shown that the lung exhibited the highest uptake of circulating [125I]-transforming growth factor-beta1 (TGF-beta1) on a per gram basis. This observation, together with the lack of information on TGF-beta receptor expression in the lung, prompted us to attempt to characterize TGF-beta receptors in this tissue. In the present report we show that the type III TGF-beta receptor is the most abundant TGF-beta binding protein in rat lung membranes and that it exhibits a 10-fold higher affinity for TGF-beta2 than for TGF-beta1. We observed that the majority of the type III receptor population in lung membranes is cleaved at a site in the central portion of the ectodomain, the resulting two fragments (95 kDa and 58 kDa) being held together by disulfide bonds. Furthermore, we demonstrate that a soluble form of the ectodomain of the type III receptor is shed from rat lung membranes in an efficient manner, with protease cleavage occurring at a site close to the transmembrane domain. This shedding is controllable by temperature, thus providing a system to study the mechanism of ectodomain release. Using this system, we show that the shedding is inhibited by prior ligand binding and by membrane solubilization. The identification of a membrane preparation which exhibits controllable and quantitative release of the type III receptor ectodomain provides a unique cell-free system for further studies of the mechanism of shedding of the type III TGF-beta receptor ectodomain.     

Concomitant loss of transforming growth factor (TGF)-beta receptor types I and II in TGF-beta-resistant cell mutants implicates both receptor types in signal transduction

A panel of 71 chemically mutagenized Mv1Lu mink lung epithelial cell clones were selected based on their resistance to the growth inhibitory action of transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2. Characterization of TGF-beta receptors in these mutants indicates that the TGF-beta-binding membrane proteoglycan, betaglycan, is apparently normal in all of them. However, 14 of the mutant clones are defective in TGF-beta receptor type I, and 22 clones are simultaneously defective in receptor types I and II. The clones with type I receptor defects fall into two distinct phenotypes, called R and LR. The R phenotype is characterized by the lack of detectable type I receptors, and has been previously described (Boyd, F. T., and Massagué, J. (1989) J. Biol. Chem. 264, 2272-2278). LR mutants are characterized by expression of low levels of type I receptor and are, like the R mutants, completely resistant to growth inhibition by TGF-beta 1 or -beta 2. Mutant clones that are simultaneously defective in receptor types I and II fall into three distinct phenotypes. These included DRa mutants which are characterized by lack of detectable receptor types I and II, DRb mutants which are characterized by low expression of both receptor types and an anomalously fast electrophoretic mobility of the type II receptor protein. All mutants that have a low level of type II receptor are also defective in type I receptor. In addition to the loss of growth inhibitory response, the receptor-defective mutants described here have lost all other responses to TGF-beta 1 and -beta 2 known to occur in parental Mv1Lu cells. The defects present in these mutant clones are not encountered in clones isolated from nonmutagenized parental Mv1Lu cells or in mutagenized cells that had not been exposed to selection with TGF-beta. The results implicate TGF-beta receptor types I and II in the mediation of a common set of cellular responses to TGF-beta. Furthermore, the high relative frequency of isolation of DR mutants raises the possibility that receptor types I and II interact as part of a common signaling TGF-beta receptor complex.     

Identification of endoglin in rat hepatic stellate cells: new insights into transforming growth factor beta receptor signaling

Transforming growth factor beta (TGF-beta) signaling is mediated by the cell surface TGF-beta type I (ALK5), type II, and the accessory type III receptors endoglin and betaglycan. Hepatic stellate cells (HSC), the most profibrogenic cell type in the liver, express ALK5, TbetaRII, and betaglycan. To monitor the expression of betaglycan in HSC, we used the commercially available antibody sc-6199 in Western blot analysis. This antibody, raised against a peptide mapping at the carboxyl terminus of the human betaglycan, is claimed to be specific for betaglycan, although it is known that the C-terminal domain is highly conserved in type III receptors. Proteins recognized in HSC by sc-6199 did not match the characteristic migration pattern of betaglycan. Moreover, the determined molecular weight (M(r) 160) and the observed reductant sensitivity after treatment with dithiothreitol resemble those of a closely related type III receptor, endoglin (CD105). Endoglin, a disulfide-linked homodimer, is an accessory component of the TGF-beta receptor complex and mainly expressed on endothelial cells. The presence of endoglin in HSC of rat liver was confirmed by molecular cloning of the endoglin cDNA and immunocytochemistry. The reactivity of sc-6199 with both auxiliary TGF-beta receptors (betaglycan and endoglin) from rats was demonstrated by Western blot and immunocytochemical analysis of cells heterologously expressing these proteins. Furthermore, Northern and Western blotting revealed that both betaglycan and endoglin genes are differentially regulated in HSC and in transdifferentiated myofibroblasts (MFB). By surface labeling and immunoprecipitation experiments, we show that endoglin is found in significant amounts exposed at the plasma membrane of HSC and MFB, which is a pivotal prerequisite for binding of and signaling in response to TGF-beta. In conclusion, we hypothesize that TGF-beta signals in HSC and MFB are tuned by two different interconnected signaling pathways, as it was previously demonstrated for endothelial cells.