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Growfli Factors, 1990, Vol. 3, pp. 35-43 Reprints available directly from the publisher Photocopying permitted by license only

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Physicochemical Activation of Recombinant Latent Transforming Growth Factor-beta's 1, 2, and 3 PETER D. BROWN't, LALAGE M. WAKEFIELD', ARTHUR D. LEVINSONZ,and MICHAEL B. SPORN', 'Laboratory of Chemopreuenhon, Building 41, Room C629, National Cancer Insfltute, Bethesda, Maryland 20892, and 'Department of Cell Genetics, Genentech lnc , 460 Point San Bruno Boulevard, South San Francisco, California 94080

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(Received September 13, 1989; Accepted 7) Native and recombinant forms of transforming growth factor-beta 1 (TGF-PI) are synthesized predominantly as biologically latent complexes. Physicochemical analysis demonstrates that the more recently described TGF-62 and TGF-63 are also latent, and reveals a common series of sharply defined parameters for activation. Human recombinant latent TGF-Ps 1 and 2 show identical profiles of activation by acid and base; the transition from latency occurs between pH 4.1 and 3.1, and between p H 11.0 and 11.9. The profile for chicken recombinant latent TGF-63 is slightly shifted with activation between pH 3.1 and 2.5, and between pH 10.0 and 12.3. Thermal activation of native and recombinant latent TGF-P1 occurs over the temperature ranges of 75-100°C and 65-100"C, respectively, with complete activation after 5 min at 80°C. Temperatures above 90°C result in thermal denaturation of TGF-61 itself. Recombinant latent TGF-Ps 2 and 3 are also activated over this temperature range; however, maximum activation occurs at 100°C. These results suggest common elements in latent complex structure despite differences between the TGF-fi subtypes in pro-region primary sequence.

KEYWORDS: transforming growth factor-beta, latent complexes, recombinant methods

INTRODUCTION

cessed precursor, a dimer termed the "latency associated peptide" (75 kD) (Wakefield et al., 1989). Transforming growth factor-beta (TGF-B) is a An additional "binding protein" (135 kD), disulfidemember of an expanding family of structurally bonded to the latency associated peptide, is present related peptides that regulate cell growth and in the native latent TGF-B1 complex obtained from function (Sporn et al., 1986, 1987; Massague, 1987). platelets and several cultured cell lines (Miyazono et The precise nature of the role of TGF-B in vivo al., 1988; Wakefield et al., 1988). remains unclear; however, much of the ongoing The latent complex of TGF-Bl from human plateresearch indicates important roles in development lets has been purified and its composition deterand in the processes of tissue repair and re- mined (Miyazono et al., 1988; Wakefield et al., 1988), modelling (Roberts and Sporn, 1989). Unlike other but little is known about the tertiary and quaternary peptide growth factors, TGF-Bl is synthesized and structure of this complex, the forces that maintain it, secreted almost exclusively as a biologically inactive and the modifications that might allow its dissocior "latent" complex (Lawrence et al., 1985; Pircher et ation. The latent TGF-B1 complex can be activated al., 1986). In recombinant TGF-B1 this complex con- by exposure to acidic or alkaline environments, by sists of the active or "mature" TGF-B1 dimer (25 kD) briefly heating at lOo"C, or by treatment with noncovalently associated with remainder of its pro- chaotropic agents (Lawrence et al., 1985; Wakefield et al., 1988). It has also been reported that the native latent TGF-Pl complex may be activated by the 'Corresponding author. limited action of proteases and glycosidases; how'Present address: Building 10, Room 2A33, Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892. ever, it appears that only a small fraction of the 35

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BROWN ct a/

complex is susceptible to this form of activation (Lyons et al., 1988; Miyazono and Heldin, 1989). The precursor pro-region sequences of TGF-/?’s 2 and 3 differ in length and show only limited homology (45-50%) to that of TGF-B1 (de Martin et al., 1987; Jakowlew et al., 1988). Since these sequences constitute the latency associated peptide, it is unclear to what extent these recently described growth factors share the latency properties of TGFB1. We demonstrate here that recombinant TGF-B2 and TGF-B3 are latent, and report the physicochemical analysis of the latency of TGF-/?’s 1, 2, and 3. Despite differences in precursor primary structure these latent complexes share common parameters of activation.

METHODS

Purification of Recombinant Latent TGF-PI The latent TGF-Bl complex in CHO cell-conditioned medium was partially purified by cation exchange and hydrophobic interaction chromatography on Mono S HR 10/10 and Alkyl Superose HR 10/10 FPLC columns (Pharmacia/LKB Biotechnologies Inc., Piscataway, NJ). Aliquots of column fractions were acidified, reneutralized, and assayed for TGF-fi using the quantitative radioreceptor assay. The peak fractions from the Alkyl Superose column were pooled and concentrated by ultrafiltration, and the ammonium sulfate removed by dialysis against phosphate-buffered saline, pH 7.4 (PBS). Glycerol was added to a final concentration of 10% prior to storage of the samples at -70°C to protect the latent complex from activation by freeze-thaw. The latent complex represented >70% of the total protein in the Alkyl Superose pool, as judged by SDS-PAGE analysis (data not shown).

Preparation of Recombinant Latent TGF-B DNA manipulation and plasmid construction was performed as described (Maniatis et al., 1982). An expression vector p(SBB) was prepared in which the coding portion of the appropriate TGF-B cDNA was inserted in the proper orientation into the vector, pRK5; this vector is designed to express heterologous genes in mammalian cells under the control of the immediate early promoter of cytomegalovirus (kindly provided by R. Klein and D. V. Goeddel). The TGF-B1 and 2 expression plasmids were transfected into CHO cells in the presence of a vector encoding the neomycin-resistance gene, pSVneoBal6 (Seeburg et al., 1984). Cells stably expressing the neo gene were then selected as described (Wakefield et al., 1989). The TGF-B3 plasmid was expressed in a transient system. CHO cells were seeded in 60mm plates at 40% confluency. The following day, the cells were transfected with 5 p g plasmid DNA, and the medium collected after 48 hr.

Activation of Latent TGF-/3

Mature TGF-B is lost rapidly from solution by adsorption to untreated plastic surfaces; in PBS or Dulbecco’s modified Eagle medium (DMEM) nearly 50% is lost within 5min and over 70% within 15min. To prevent such losses, all tubes were siliconized with Sigmacote (Sigma Chemical Company, St. Louis, MO) and bovine serum albumin (1-0.2 mg/ml) was included in all activation assays. Commercially available polypropylene tubes impregnated with silicone were found to be far less effective in preventing losses. Total TGF-B activity was determined by transient acidification of samples with 5 M HCl to pH 2.0-2.5 for 10 min at room temperature, followed by reneutralization to pH 7.5 with 5 M NaOH and 1M HEPES buffer, pH 7.4. For activation by pH, aliquots (1Opl) of latent TGF-B were added to 500 pl 0.05 M citric acid/phosphate buffer, pH 2.5-7.1, or glycine/NaOH buffer, pH 8.3-12.3. After 30 min at room temperature, the Preparation of Platelet Latent TGF-B1 samples were diluted 100-fold and assayed for TGFFresh human platelets were induced to degranulate B activity by the CC1-64 growth inhibition assay (see and a platelet secretate obtained as previously below). For thermal activation, aliquots of latent described (Wakefield et al., 1988). This secretate TGF-P in PBS were heated in water baths for typically contained 50-200 n g / d of TGF-B1 and 1-10 min and then immediately cooled in ice water. 200-300 pg/ml protein. The secretate was further Samples were then assayed for TGF-B activity as enriched 30-fold for TGF-B by Mono Q anion- described. exchange chromatography, the latent complex Aliquots of recombinant latent TGF-B1 were also eluting as a broad peak between 0.34 and 0 . 4 4 ~ examined for activation by treatment with plasmin NaCl (Wakefield et al., 1988). (Boehringer Mannheim Biochemicals, Indianapolis,

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A C l l V A l I O N Of- LATFNT TGF-P

37

IN), neuraminidase (high-purity type X, Sigma), and N-glycanase (Genzyme Corporation, Boston, MA). Incubations with plasmin were performed for 2 hr at 37°C in PBS. The serine protease inhibitor, aprotinin, was then added, and the samples were diluted 50-fold and assayed for TGF-B activity as described. Incubations with neuraminidase and Nglycanase were performed for 18 hr at 37°C in 0.05 M citric acid/phosphate buffer, pH 6.1, and PBS, respectively.

TGF-@was demonstrated by the full reversal of any inhibitory activity by the addition of a polyclonal anti-TGF-B antibody (R&D Systems Inc., Minneapolis, MN). All samples were assayed at approximately 5 p TGF-P, ~ which is just subsaturating for this assay when [3H]thymidine is used as a label. A radioreceptor assay was used to assay active TGF-P in samples of latent TGF-Bl treated with plasmin (O'Connor-McCourt and Wakefield, 1987).

CCL-64 Growth Inhibition Assay

RESULTS

TGF-B activity was assayed by measuring the inhibition of growth of the mink lung cell line, CCI64 (American Type Culture Collection, Rockville, MD). Growth inhibition was measured as the decrease in incorporation of ['HI thymidine as described (Danielpour et al., 198Y). Specificity for

Activation of Latent TGF-P by pH A profile of activity over a pH range of 2.5-12.3 was constructed for each of the forms of latent TGF-P (Fig. 1).The transition points for each profile have been confirmed in repeat experiments and the

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PH FIGURE 1. Profiles of pF1 activation of re(-ombinant latent TGF-PI (A), recombinant latent TGF-P2 (B), recombinant latent 'TGF-P3 (C), and native platelet latent TGF-PI (D). TGF-/3 activity was determined by the CCL-64 growth-inhibition assay a s described. Vertical broken lines at pH 3.1 and 11.9 mark the values at which recombinant latent TGF-Pl and 2 are fully activated. Data points are means of duplicate values.

BROWN et al

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accuracy of the pH values checked. The profiles for recombinant latent TGF-B1 and 2 were very similar, with sharp transitions from latency occurring between pH 4.1 and 3.1 and between pH 11.0 and 11.9 (Fig. lA, B). Limited activation at pH 4-5 has been reported previously (Lyons et al., 1988; Lawrence et al., 1985); however, no activation over this mildly acidic pH range was observed in any of the experiments performed for the current study. The activation profile for recombinant latent TGFp 3 differed from that of TGF-B1 or 2 (Fig. 1C). The transitions from latency occurred between pH 3.1 and 2.5, and between pH 10.0 and 12.3. Native latent TGF-B1, from human platelets, was also found to be more stable at pH 3.1 than its recombinant counterpart, but the transition from latency between pH 11.0 and 11.9 was equally as sharp (Fig. 1D).

Thermal Activation of Latent TGF-B The thermal activation of recombinant latent TGFp1 was studied by incubating aliquots of latent TGF-pl for 1, 5, and 10min over a temperature range of 60-100°C (Fig. 2). Complete activation was observed after 10 min at 70"C, 5min at 75"C, and 1 rnin at 85-90°C. When mature TGF-pl was incubated over this temperature range for 10 min, inactivation was observed at temperatures above 80°C (Fig. 2, inset). This thermal denaturation of mature TGF-pl explains the decrease in TGF-p activity observed in the 10 min profile of the latent complex above 80°C. A comparison was made of the thermal stability of the different latent forms by heating for 5min over the same range of temperatures. Again, recombinant latent TGF-pl and 2 showed similar

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FlGURE 2. Time dependence of thermal activation of recombinant latent TGF-Bl. TGF-B activity, as determined by the CC1-64 growth-inhibition assay, was measured after heating recombinant latent TGF-B1 for 1 (A), 5 (O),and 10 min (0). The "inactivation"of mature TGF-PI is shown for a 10-min incubation over the same temperature range (inset). Data points are means of duplicate values.

profiles of activation, although latent TGF-PZ was more stable at 100°C (Fig. 3A, B). Native latent TGFpl was more stable than recombinant latent TGF-P1 and showed a very sharp transition from latency between 75 and 80°C (Fig. 3A). Recombinant latent TGF-B3 differed slightly from the other latent forms and showed increasing degrees of activation from 60-100°C (Fig. 3B).

described, 12-18O/0 of acid-activated TGF-P1 in a 5 0 O p ~solution reformed a latent complex over a 10 min incubation period at room temperature. Under the same conditions, no reassociation of thermally activated TGF-P1 was detectable. Immediate dilution and assay of acid- and thermally activated TGF-Pl also showed that the latter liberated approximately 1.2-fold greater activity. This implies

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Temperature (" Cl FlGURE 3. Thermal activation of (A) recombinant ( e )and native ( 0 )latent TGF-PI and (B) recombinant latent TGF-P2 (A)and recombinant latent TGF-B3 ( A ) . Samples were heated for 5 min over the range of temperatures shown, and TGF-B activity was determined by the CCL-64 growth-inhibition assay A vertical line at 75°C is drawn a s a point of reference Samples of latent TGF-j3 were also acid-activated as described in Methods and assayed for TGF-b activity (AA). Data points are means of duplicate values.

Interestingly, the total TGF-Pl activity as determined by acid activation was found to be slightly less than the maximum activity obtained by thermal activation (Fig. 3). Subsequent experiments revealed that the reassociation of the latent complex following acidification and reneutralization, as recently reported (Wakefield et al., 2989), does not occur to the same extent following thermal activation. Indeed, under the experimental conditions

a degree of heterogeneity in the sample of latent TGF-Pl, although the structural basis for this has not been determined. For these reasons, thermal activation may be the method of choice for activating latent TGF-/3 in conditioned media. Acid activation of recombinant forms of latent TGF-P1, 2, and 3 was also carried out at pH 2.0 and 1.2. These conditions failed to yield greater activity than the standard acid-activation procedure, and

BROWN et a/

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again yielded slightly less activity than thermally activated samples in the same experiment (data not shown).

molecules in the sample. Latent TGF-Pl was incubated with 0-1 U/ml plasmin for 2 hr at 37"C, and the activity was compared with total TGF-81 activity as determined by acid activation (Fig. 4). Although 1U/ml did appear to liberate some mature Activation of Latent TGF-/3by Limited Proteolysis TGF-P1, it is clear from the total activity at this conand Glycolysis centration of protease that substantial inactivation of Since it has been reported widely in the literature mature TGF-Pl itself also occurred. In a duplicate that the limited action of proteases or glycosidases experiment, inactivation was observed at concenresults in the partial activation of native latent TGF- trations of 0.3U/ml. Activation of latent TGF-Bl at 81 (Lyons et al., 1988; Miyazono and Heldin, 1989), 1U/ml plasmin was also appreciably less prorecombinant latent TGF-Bl was tested for suscepti- nounced than in the experiment shown. In a separate experiment, the ability of neuraminibility to such action. The activation of recombinant latent TGF-Pl by plasmin was studied using A549 dase and N-glycanase to activate recombinant latent cells in a radioreceptor assay. Although not as sensi- TGF-Pl was examined in the more sensitive CCL-64 tive as the CCL-64 growth-inhibition assay, the growth-inhibition assay (Figure 5). Full reversal of radioreceptor assay is less affected by other the inhibitory activity liberated by heat and acid

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Plasmin (U/ml) FlGURE 4. Activation and subsequent degradation of recombinant latent TGF-PI by plasmin. Samples of latent TGF-PI were incubated with plasmin at the concentrations shown for 2 hr at 37°C. Active TGF-PI was then determined in neutral samples (0),and total TGF-PI in acid-activated samples (O),by the radioreceptor assay. The amount of TGF-PI was determined from dilution curves from each value of plasmin concentration. Total (acid-activation) TGF-Bl data points are means of duplicate values.

ACTIVATION OF LATFNT

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treatment was obtained with the addition of antiTGF-P antibody, confirming that this inhibition was due to active TGF-B. Indeed, treatment of cells with this antibody caused increased cell growth relative to untreated control cells, most probably as a result of blocking the growth inhibition caused by small amounts of TGF-B present in 0.2'70 (vol/vol) fetal bovine serum or secreted by CCL-64 cells. The addition of untreated latent TGF-P1 to the assay caused a 10% inhibition of cell growth, a reflection of the 2-3% active fraction in the latent preparation. When latent TGF-P1 was enzymatically pretreated, limited activation was seen with neuraminidase. However, this activity represents only 10-lSo/~ of the total TGF-B1 and may be due to the activation of an altered subfraction of the latent complex. It should also be noted that although high-purity preparations of glycosidases were used, the action of contaminating proteases cannot be ruled out. Treatment of native platelet latent TGF-B1 with plasmin and the two glycosidases yielded very similar results.

DISCUSSION The experiments described have established that the genes for TGF-P2 and TGF-P3, like that for TGF-Pl, specify the synthesis of biologically latent forms of these peptides. The pH and temperature at which these different latent TGF-B complexes become active have been studied. In this respect, it has been

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FIGURE 5 Activation of recombinant latent TGF-PI by glycosidases. Samples of latent TGF-Dl were incubated with neuraminindase (NA) or N-glucanase (NG) for 18 hr at 37°C. Both enzymes were used at a final concentration of 1 U/ml. Samples of latent TGFPI were also activated by acid treatment as described and by heating at 80°C for '1 min. An untreated sample of latent TGF-Bl is shown as a control. TGF-P activity was determined in the absence (solid bars) or presence (hatched bars) of anti-TGF-P antibody by the CCL-64 growth-inhibition assay. A decrease in ('Hlthymidine incorporation represents the liberation of active TGF-P. Data are means ot duplicate values.

demonstrated that although there is only limited sequence homology in the latency associated peptides of TGF-Ps 1, 2, and 3, the latent growth factor complexes are remarkably similar in their activation profiles. The pH profiles in the current study show no evidence of activation under mildly acidic conditions (pH 4-5) for any of the latent forms tested. This finding differs from earlier reports of the activation of latent TGF-P in fibroblast-conditioned medium (Lawrence et al., 1985; Lyons et al., 1988). In these studies, a small fraction of the total TGF-/? was found to be active over the range of pH 4-5. However, in each study the conditioned medium was adjusted to the appropriate pH by the direct addition of HCl. Since the dissociation of the latent complex at low pH is rapid (unpublished observation), the direct addition of acid may cause a transient local drop in pH to below pH 4, thus activating a small fraction of the latent form. In a separate study of the activation by pH of purified human platelet latent TGF-B1, no activation was observed above pH 3.5 (Miyazono et al., 1988). As in the current study, pH adjustments were made by the addition of small aliquots of latent TGF-B to larger volumes of buffers of predetermined pH. Although it is possible that the latent form of TGF-B in fibroblastconditioned medium is different from any of the forms tested here, it seems unlikely that latent TGFPs 1, 2, and 3 are significantly activated above pH 3.1-3.5.

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BROWN et al

This has important implications since it has been proposed that acidic microenvironments that exist around specific cell types might activate the latent complex in vivo (Roberts and Sporn, 1989). In recent studies with microelectrodes it has been shown that osteoclasts and macrophages can generate acidic microenvironments as low as pH 3.0 and 3.6, respectively (Silver et al., 1988). Indeed, activated osteoclasts have recently been shown to activate latent TGF-#? in vitro (Oreffo et al., 1989). Under these conditions it is possible that the different subtypes of native latent TGF-#? may be activated selectively: Recombinant latent TGF-#?3 remains latent pH 3.1, while recombinant latent T G F - ~ s1 and 2 are active. Thermal activation of the latent TGF-B complexes proved to be an effective means of liberating mature TGF-B, consistently yielding slightly greater activity than acid activation. While this method of activation is unlikely to have physiological significance, the similarity of the thermal activation profiles again suggests that similar forces hold the complexes together. Importantly, from an experimental standpoint, the rate of reassociation of the latent TGF-B1 complex after heating was reduced relative to that following acid activation. This reduces the variable of reassociation during sample preparation and has led to more reproducible radioreceptor and growthinhibition assays. The lower rate of reassociation may be due to the thermal denaturation of the 75kD latency associated peptide, which has a lower cysteine content than mature TGF-B. The results of studies on the activation of latent TGF-#?1 by plasmin and neuraminidase support recent findings by others of partial activation (Lyons et al., 1988; Miyazono and Heldin, 1989). In each of these studies, activation was limited to a maximum of 15-209'0 of the available TGF-#?1, even in the presence of relatively high levels of enzyme activity. It therefore seems unlikely that such mechanisms constitute the principal means by which cells activate TGF-B1 in vim, especially in light of the fact that levels of plasmin that liberate active TGF-Pl also cause substantial inactivation of the same. Although it is possible that a single unidentified enzyme could be responsible for activation in vivo, it now seems more likely that the mechanism is more complex, possibly a multistep process involving complementary actions of different cell types. This is suggested by a recent report in which it was shown that pericytes and capillary endothelial cells each secrete latent TGF-B when cultured separately but

secrete or generate active TGF-#?when cocultured (Antonelli-Orlidge et al., 1989). It is probably through the study of such systems that physiologically relevant mechanisms of activation will first be elucidated. The molecular basis for the minor differences in acid/alkali lability and thermal stability of the four forms of latent TGF-#?studied has not been determined; however, it is likely that the presence or absence of the 135-kD binding protein and the extent of glycosylation are important determining factors. The native TGF-P1 complex, which contains the binding protein, was more stable to both heat and acid than the recombinant TGF-#?l complex which lacks this component. Most importantly, the studies presented here have revealed a marked similarity in the properties of the latent complexes of the different TGF-B subtypes. This strongly suggests that key elements of latency may lie in one or more of the conserved regions in the primary sequence of the precursor pro-region that forms the latency associated peptide. These regions should therefore be the focus of future studies.

ACKNOWLEDGMENTS We thank Diane Smith and Christine Naugle for their technical assistance.

REFERENCES Antonelli-Orlidge, A,, Saunders, K . B., Smith, S. R. and dAmore, P. A. (1989) An activated form of transforming growth factor b is produced by cocultures of endothelial cells and pericytes. Proc. Natl. Acad. Sci. USA 86, 4544-4548. Danielpour, D., Dart, L. L., Flanders, K. C., Roberts, A. 8. and Sporn, M. B. (1989) Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-PI and TGF-p2) secreted by cells in culture. I. Cell. Physiol. 138, 79-86. de Martin, R., Haendler, B., Hofer-Warbinek, R., Gaugitsch, H., Wrann, M., Schlusener, H., Seifert, J. M., Bodmer, S., Fontana, A. and Hofer, E. (1987) Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-B gene family. EMBO J. 6, 3673-3677. tkowlew, S. B., Dillard, P. J . , Kondaiah, P., Sporn, M. B. and Roberts, A. B. (1988) Complementary deoxyribonucleic acid cloning of a novel transforming growth factor-B messenger ribonucleic acid from chick embryo chondrocytes. Mol. Endocrinol. 2, 747-755. ,awrence, D. A,, Pircher, R. and Jullien, P. (1985) Conversion of a high molecular weight latent P T G F from chicken embryo fibroblasts into a low molecular weight active /?-TGF under acidic conditions. Biochem. Biophys. Res. Commun. 133, 1026-1034.

Lyons, R. M., Keski-Oja, J. and Moses, H. I.. (1988) Proteolytic activation of latent transforming growth factor-p from fibroblast conditioned medium / Cdl Ricil. 106, 1659-lh65. Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982) Moiecular Cloriirig. A Laboratory Mariiiul, Cold Spring Harbor Laboratory,

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Massague, J . (3987) The TGF-D family of growth and differentiation factors. Cell 49, 437-4321 Miyazono, K and Heldin, C-H (1989) Role t w carbohydrate structures in TGF-Dl latency. Naturr338, 158-160. Miyarono, K., Hellman, U , Wernstedt, C. and Heldin, C-El. (1988) Latent high molecular weight complex of transforming growth factor B1. /. Biol. Clicni. 263, 6407-6415. O’Connor-McCourt, M. D. and Wakefield, L. M. (1987) Latent transforming growth factor-D in serum; a specitic complex with alpha,-macroglobulin. /. Biol. Clieni. 262, 141J90-14099. Oreffo. R. 0. C., Mundy, G R . , Seyedin, S M and Bonewald, L. M. (1989) Activation of the bone-derived latent TGF beta complex by isolated osteoclasts. Biocfierrr. Biopliys. Res. Conimun. 158, 817-823. I’ircher, R., Jullien, I’ and Lawrence, D. A . (1986) BJransforming growth factor is stored in human blood platelets as a latent high molecular weight complex Bioclrcnr. Biopliys. Res. Cornmuti. 136, 30-37. Roberts, A. 8. and Sporn, M. B. (1989) in I’eptidr2 Growth Factors and Tlierr Receptors, M. B. Sporn and A . B. Roberts, eds. (in press). Roberts. A . B., Sporn, M. B.. Assoian, R K , Smith, J. M., Roche, N S., Wakefield, L. M., Heine, U. I., Liotta, L. A,, Falanga, V., Kehrl, R. H. and Fauci, A S. (1986) Transforming growth factor type-beta: rapid induction of fibrosis and angiogenesis in 7’17’0 and stimulation of collagen formation i r i rvtro. Proc. Natl. Acad. Sci. USA 83, 1167-4171

Roberts, A. B., Flanders, K. C., Kondaiah, P., Thompson, N . L., Van Obberghen-Schilling, E., Wakefield, L. M., Rossi, P., De Crombrugghe, B , Heine, U. I . and Sporn, M. B. (1988) Transforming growth factor B: biochemistry and roles in embryogenesis, tissue repair and remodeling, and carcinogenesis. Rcccnt Prog Horni Res. 44, 157-197 Seeburg, 1’. H., Colby, W. W., Capon, D. J., Goeddel, D. V. and Levinson, A. D. (1981) Biological properties of c-Ha-ras I genes mutated at codon 12. Nuture312, 71-75. Silver, 1. A,, Murrills, R . J. and Ftherington, D. J . (1988) Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp. Cell Res. 175, 266-267. Sporn, M. B., Roberts, A . B., Wakefield, I.. M. and Assoian, R. K. (1986) Transforming growth factor-B: Biological function and chemical structure. Science 233, 532-534. Sporn, M. B., Roberts, A . B., Wakefield, L. M. and de Crombrugghe, 8. (1987) Some recent advances in the chemistry and biology of transforming growth factor-beta. /. Cell Biol. 105, 1039-1045. Wakefield, L M I Smith, D. M., Masui, T., Harris, C C and Sporn, M. B. (1987) Distribution and modulation of the cellular receptor for transforming growth factor-B. /. Cell B i d . 105, 965-975. Wakefield, L. M., Smith, D. M., Flanders, K. C. and Sporn, M. B. (1988) Latent transforming growth factor-B from human platelets: a high molecular weight complex containing precursor sequences. /. B i d . Clieni. 263, 7616-7651. Wakefield, L . M I Smith, D. M., Bro7, S Jackson, M., l.evinson, A . D and Sporn, M. B. (19219) liecombinant IGF-DI is synthesized as a two-component latent complex that share5 some structural teaturcs with the iiativt. platelet latent TCF-Pl complex. Groic$li Fnstorz 1, 203-21 21

Physicochemical activation of recombinant latent transforming growth factor-beta's 1, 2, and 3.

Native and recombinant forms of transforming growth factor-beta 1 (TGF-beta 1) are synthesized predominantly as biologically latent complexes. Physico...
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