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production of chrondrocyte-specific proteoglycan with EDs0 values of approximately 0.5-1 ng/ml in the DHCB assay.
Acknowledgments We thank Dr. Hans Marquardt for sequencing bovine TGF-/32, David Schmidt for purification and characterization of TGF-/31 and TGF-/32 from bovine bones, and Andrea Thompson for comments on the description of the chondrogenesis assays.
[31] Identification and Activation of Latent Transforming Growth Factor [3
By DAVID A. LAWRENCE Introduction Transforming growth factor/3 (TGF-/3) has been the subject of several recent reviews, 1.2so here only some of its salient properties will be recalled as an anchor point for this chapter, which is specifically concerned with latent TGF-/3 (L-TGF-/3). In its active form TGF-/3 is multifunctional and can stimulate or inhibit cell proliferation, cell differentiation, and cellular function depending on cell type (e.g., fibroblast or epithelial cell) and on what other growth factors are present in the local environment. Most of our knowledge about the biological effects of TGF-/3 concern TGF-/31, simply because the other isoforms TGF-/32, -/33, and -/34 were discovered more recently. 3-7 TGF-/31 is produced by most cell types s and, unlike 1 M. B. Sporn, A. B. Roberts, L. M. Wakefield, and B. de Crombrugge, J. Cell Biol. 105, 1039 (1987). 2 D. A. Lawrence, "Malignant Cell Secretion" (V. Krsmanovic, ed.), p. 215. CRC Press, Boca Raton, Florida, 1990. 3 S. Cheifetz, J. A. Weatherbee, M. L. S. Tsang, J. K. Anderson, J. E. Mole, R. Lucas, and J. Massagu6, Cell (Cambridge, Mass.) 48, 409 (1987). 4 T. lkeda, M. N. Liobin, and H. Marquardt, Biochemistry 26, 2406 (1987). 5 p. Ten Dijke, P. Hansen, K. K. lwata, C. Pieler, and J. G. Foulkes, Proe. Natl. Acad. Sci. U.S.A. 85, 4715 (1988). 6 S. Jakowlew, P. J. Dillard, P. Kondaiah, M. B. Sporn, and A. B. Roberts, Mol. Endocrinol. 28, 747 (1988). 7 R. Derynck, P. B. Lindquist, A. Lee, D. Wen, J. Tamm, J. L. Graycar, L. Rhee, A. J. Mason, D. A. Miller, R. J. Coffey, H. L. Moses, and E. Y. Chen, EMBO J. 7, 3737 (1988). 8 R. Derynck, J. A. Jarrett, E. Y. Chert, D. H. Eaton, J. R. Bell, R. K. Assoian, A. B. Roberts, M. B. Sporn, and D. V. Goeddel, Nature (London) 316, 701 (1985).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright ct~ 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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classic hormones, acts locally via paracrine and autocrine modes.9, ~0However, before such local action can occur, TGF-/31 must first be activated, as most of this growth regulatory molecule is released from producer cells in an inactive, latent form. ~1-14 Because TGF-fi is resistant to acid pH (pH 2.0), 15'16 purification schemes developed for TGF-/3 made full use of acetic acid for dialysis of cell-conditioned media and cell extracts and as eluant for gel-filtration columns. 17 Purification under acid conditions always yielded the biologically active 25-kDa dimer, by dissociation of the latent complex (see later). Thus, the latency phenomenon had been completely overlooked until this laboratory reported that normal chicken, mouse, and human embryo fibroblasts all released a TGF-/3 activity in a latent form under neutral conditions. I~ Quite soon after we showed that other fibroblasts, normal and transformed by various RNA and DNA viruses, also secreted L-TGF/3.12"~8Perhaps more important was the demonstration ~4that human blood platelets (with bone one of the richest sources of TGF-/319) contained a latent form of TGF-/3. Gel-filtration runs performed during the initial studies indicated that L-TGF-/3 was of high molecular weight (>400K).11'13'14 Considering the activation treatments (see later) it was proposed that LTGF-/3 was a complex of 25-kDa TGF-fl associated noncovalently with a carrier protein. ~3 Moreover, it was suggested that activation of L-TGF-/3 could be a critical step in the regulation of biological activity. 14 As a general phenomenon, the latency of TGF-/31 has been confirmed by several laboratories. 20,21 9 M. B. Sporn, A. B. Roberts, L. M. Wakefield, and R. K. Assoian, Science 233, 532 (1986). J0 A. S. Goustin, E. B. Leof, G. D. Shipley, and H. L. Moses, CancerRes. 46, 1015 (1986). tl D. A. Lawrence, R. Pircher, C. Kryc~ve-Martinerie, and P. Jullien, J. Cell. Physiol. 121, 184 (1984). J2 R. Pitcher, D. A. Lawrence, and P. Jullien, Cancer Res. 44, 5538 (1984). 13 D. A. Lawrence, R. Pitcher, and P. Jullien, Biochem. Biophys. Res. Commun. 133, 1026 (1985). 14 R. Pitcher, P. Jullien, and D. A. Lawrence, Biochem. Biophys. Res. Commun. 136, 30 (1986). 15 H. L. Moses, E. L. Branum, J. A. Proper, and R. A. Robinson, Cancer Res. 41, 2842 (1981). 16 A. B. Roberts, M. A. Anzano, L. C. Lamb, J. M. Smith, and M. B. Sporn, Proc. Natl. Acad. Sci, U.S.A. 78, 5339 (1981). 17 M. B. Sporn, M. A. Anzano, R. K. Assoian, J. E. De Larco, C. A. Frolik, C. A. Meyers, and A. B. Roberts, Cancer Cells 1, l (1984). 18 C. Kryc~ve-Martinerie, D. A. Lawrence, J. Crochet, P. Jullien, and P. Vigier, Int. J. Cancer 35, 553 (1985). t9 j. L. Carrington, A. B. Roberts, K. C. Flanders, N. S. Roche, and A. H. Reddi, J. Cell Biol. 107,1969 (1988). 2o R. J. Coffey, G. D. Shipley, and H. L. Moses, Cancer Res. 46, 1164 (1986). 2J L. M. Wakefield, D. M. Smith, T. Masui, C. C. Harris, and M. B. Sporn, J. Cell. Biol. 105, 965 (1987).
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Sample Preparation
Latent Transforming Growth Factor fi Many cell types are still cultured in the presence of serum, despite the advancing knowledge regarding growth factor supplements 2z to minimal media containing salts, amino acids, and vitamins. In vivo, however, cells only come into contact with serum at wound sites. 22A further disadvantage of using serum in the present context is that it contains numerous growth factors in variable quantities, 22 including TGF-fl essentially in a latent form. 23"24 Where no satisfactory serum-free medium has been found in which the desired cells grow, one can often avoid the problem by first growing the cells in medium with serum and then washing the cells, at least twice, with serum-free medium and reincubating them for 12 to 48 hr in the serum-free medium, which is then harvested to provide a virtually serum-free cell-conditioned medium. During the washing procedure it is important to incline the culture dish and drain it to remove as much of the initial serum-containing growth medium as possible. Some investigators reincubate the washed cultures with serum-free medium for 6-24 hr and then discard the latter before further incubation in serum-free medium, which will constitute the sample for testing. 2°'2~ This additional step removes further serum components. Clearly, each investigator must determine the culture requirements of cells used, but where serum must be used this does not necessarily preclude identifying TGF-fl in such samples. Many growth factors, including TGF-fl, can be expected to accumulate in culture medium with time; harvesting after a 24-hr period is common, and, where cell viability is maintained, repeated harvests over 4-5 days can be made. It is possible to identify activatable TGF-fl from only 1.0 ml of a crude, unconcentrated sample, but using 5.0 ml or more is simpler at the bench level. After harvesting, the sample should be centrifuged (10 min, 4000 g) to remove whole cells and most debris. Depending on the volume available, the clarified conditioned medium can be divided into 2.0- to 5.0-ml aliquots, some of which can be stocked at - 2 0 ° or below for later use. One should avoid defrosting a sample more than once, as it has been reported that repeated freezing and thawing tend to activate L-TGF-fi. 25In contrast, purified TGF-fl (25 kDa) tends to be inactivated by this treatment.
z2 D. Barnes and G. Sato, Cell (Cambridge, Mass.) 22, 649 (1980). 2s C. Bjornson-Childs, J. A. Proper, R. F. Tucker, and H. L. Moses, Proc. Natl. Acad. Sci. U.S.A. 79, 5312 (1982). 24 M. O'Connor-McCourt and L. M. Wakefield, J. Biol. Chem. 262, 14090 (1987). _~5R. M. Lyons, J. Keski-Oja, and H. L. Moses, J. Cell Biol. 106, 1659 (1988).
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Latent Transforming Growth Factor fl Neutral, nondissociating conditions must be used to prepare samples, otherwise some activation will occur. Sonication and/or homogenization (e.g., with a Dounce-type homogenizer) are convenient for preparing extracts of most cells. We have used sonication (30-sec duration at 8/zm peak to peak with a 1.0 cm diameter probe) to prepare L-TGF-fl from human blood platelets, j4 Washed platelets are taken up in phosphatebuffered saline (PBS), or other neutral buffer (1 mg wet weight/20 mi). After sonication the homogenate is centrifuged (100,000 g for 1 hr) and the resulting supernatant taken as the neutral extract. For tissue extraction the reader is referred to standard procedures for tissue maceration, bearing in mind the necessity of using neutral buffers without detergents.
Activation of Latent Transforming Growth Factor fil The most frequently used activation method is acidification, usually followed by reneutralization to render the sample biocompatible again.mJ It is convenient to prepare the following solutions: 1.0, 0.5, and 0.1 M HCI and NaOH in culture-grade, distilled water. Depending on the sample volume and the buffering capacity of its contents (e.g., serum or HEPES buffer will provide extra buffering strength), HCI is added dropwise with rapid mixing until pH 2.0 is obtained. Approximately 100/zl of 1.0 M HCI added to 2.0 ml Dulbecco's medium leads to pH 2.0. At this low pH all L-TGF-fll is activated (e.g., a 20-fold increase in activity over an original sample of conditioned medium 13 and a 300-fold increase for an acidified human platelet extract compared to the neutral extractl4), but considerable activation occurs (-7-fold) when chicken embryo fibroblast-conditioned medium, which contains latent TGF-fl, is brought to pH 5.0.13 To avoid excessive dilution during acidification, sample volumes greater than 10.0 ml should be treated with concentrated HCI (-10.0 M). The acidified sample is left for 1 hr at 4 °. (These were the original conditions for standardizing the procedure. Because activation appears to be due to pH shock dissociating the latent complex, the above time might well be shortened to about 10 min as long as this practice is followed throughout.) Reneutralization of the acidified sample is carried out by the dropwise addition of NaOH to return to the initial pH. Because pH can rise above pH 8.0, care must be exercised and use made of lower NaOH molarities to obtain the final pH. The alkaline overshoots are probably of little consequence as alkalinization is another activation treatment for L-TGF-fl. 13
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Comments. The protein concentration of serum-free conditioned medium can be quite variable depending on cell type and culture conditions, but it rarely exceeds 100 /xg/ml. Acidification of such media does not normally lead to visible turbidity unless serum is present. Such precipitated material can distort assays of activated samples and should be removed by centrifugation (10 min, 4000 g). Some microbial contamination of samples undergoing activation will inevitably occur during pH meter manipulations, but this can be cut to a minimum by using sterile pipettes, micropipette cones, and solutions of acid and base. To avoid potential losses of protein by filtration (e.g., 0.45-/xm cartridges) one may sterilize the samples for bioassay by 6°Co-irradiation, although such facilities are not generally available, leaving filtration as the more common method for sample asepsis. With an activated and sterile sample in hand, one can proceed to the actual assay of TGF-/3. The investigator can choose one or more of these standard assays: stimulation of colony formation of NRK-49F fibroblasts in soft agar in the presence of epidermal growth factor (EGF)26;inhibition of [3H]thymidine incorporation in certain sensitive cell lines, like the mink epithelial cell line CCL6427;radioreceptor competition assay28'29; and neutralization or Western blotting with specific anti-TGF-/3 antiserum. 3°,3j The first three assays can be used with appropriate standards of known TGF-/3 concentrations to determine less than 1.0 ng/ml TGF- 3. As a control for activated samples of TGF-/3 one should check, in addition to the original sample, an aliquot which received premixed amounts of acid and base (this sample ought to give the same result as the original unactivated sample). For assays on conditioned media, one should test an aliquot of culture medium that has not been in contact with the cells. One-half of the aliquot should be tested directly and the other half should receive the acid/base treatment. Secretion of spontaneously active L-TGF-/31 appears to be relatively uncommon. For large samples needing further purification, another convenient acid-activating treatment, is dialysis in acetic acid. Both I% and I M 26 j. E. De Larco and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 75, 4001 (1978). 27 R. F. Tucker, G. D. Sfiipley, H. L. Moses, and R. W. Holley, Science 226, 705 (1984). 2~ C. A. Frolik, L. M. Wakefield, D. M. Smith, and M. B. Sporn, J. Biol. Chem. 259, 10995 (1984). 29 R. F. Tucker, E. L. Branum, G. D. Shipley, R. J. Ryan, and H. L. Moses, Proc. Natl. Acad. Sci. U.S.A. 81, 6757 (1984). 30 T. Masui, L. M. Wakefield, J. F. Lechner, M. A. LaVeck, M. B. Sporn, and C. C. Harris, Proc. Natl. Acad. Sci. U.S.A. 83, 2438 (1986). st L. M. Wakefield, D. M. Smith, K. C. Flanders, and M. B. Sporn, J. Biol. Chem. 263, 7646 (1988).
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(5.7%) acetic acid solutions can be employed. Acetic acid (1 M) gives a pH of 2.4, low enough to activate all L-TGF-/3. As acetic acid is evaporated during lyophilization, samples thus treated can be reconstituted in the desired buffer or culture medium and also concentrated at the same time by appropriate volume reduction. Other Methods of Activating Latent Transforming Growth Factor/31
Boiling Boiling is a convenient procedure for 20 or more samples (e.g., column fractions or conditioned media from several cell types or from various culture conditions) where acidification/neutralization becomes tedious. The sample is kept in boiling water for 3 min. This heat treatment is about 50% less effective than acidification. ~3Human platelet L-TGF-/31 cannot be activated by boiling, 3z whereas that of rat platelets can be activated in this way. 33
Alkalinization Alkalinization is simply the reverse of the acidification procedure. The pH of the sample is brought to pH 11.0 or more by dropwise addition of NaOH before reneutralization with HCI.
Urea Exposure of the sample to 8 M urea at room temperature activates LTGF-/31 but tends to be a less effective method for L-TGF-/31 in conditioned media 13 and platelet extracts.14
Enzymatic Activation By Plasmin. A 2-hr incubation at 37° with 0.2 U plasmin per milliliter conditioned medium gives slightly less than one-half the activation obtained by acidifying to pH 1.5 (respectively 450 and 1000 colonies in the soft agar assay).25 The plasmin treatment is stopped by adding phenylmethylsulfonyl fluoride and aprotonin at 2.0 mM and 0.55 TIU/ml final concentrations followed by 1.0 mg bovine serum albumin (as a carrier) per milliliter of reaction mixture. In the TGF-/3 radioreceptor assay, maximum inhibition is obtained following treatment with 0.5 U/ml plasmin. Evidence 32 K. Miyazano, U. Hellman, C. Wernstedt, and C. H. Heldin, J. Biol. Chem. 263, 6407 (1988). 33 T. N a k a m u r a , T. Kitazawa, and A. lchihara, Biochem. Biophys. Res. Commun. 141, 176 (1986).
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is provided 25 for the existence of two pools of L-TGF-/3, one being activated by plasmin (or mild acid pH 4.5) and the other by strong acid at pH 1.5. Further evidence for plasmin activation comes from coculture experiments. 34 Here, the migration of confluent bovine aortic endothelial cells into a "wounded area" (obtained by scraping across an adherent cell monolayer in a petri dish with a razor blade) is blocked by cocultivating bovine pericytes or smooth muscle cells in the denuded area. This blockage appears to be due to plasmin-mediated activation of L-TGF-/3 in situ. By Endoglycosidase F. Endoglycosidase F (endo F) removes complex and oligomannose (high mannose) N-linked carbohydrates. 35-37L-TGF-/31 purified from human platelets (see below) is incubated at 37 ° for 16 hr in 50.0 mM EDTA and 100.0 mM sodium phosphate, pH 7.4, containing 10 or 25 U of endo F from Flavobacterium meningosepticum (Boehringer, Mannheim, Germany) per milliliter of reaction mixture. 38 In a radioreceptor assay, 0.5 nM (purified) L-TGF-/31 pretreated with 10 or 25 U/ml of endo F gives the same degree of inhibition as does 0.1 nM L-TGF-/31 transiently acidified to pH 2.0. By Sialidase (N-Acetylneuraminidase). Several enzymes have the general name sialidase; depending on their specificity, they break particular linkages to remove N-acetylneuraminic acid from various polysaccharides, glycoproteins, and gangliosides. 36.37The sialidase used in the report 38 cited here is type V from CIostridium perfringens (Sigma, St. Louis, MO). Purified L-TGF-/31 at 320 pM is incubated with 2 U of sialidase per milliliter of reaction mixture in 100 mM sodium phosphate buffer, pH 5.6, for 18 hr at 37°. The effect of the treatment is assayed by the inhibition of [3H]thymidine incorporation; the extent of inhibition is about 80%. Even with saturating concentrations of sialidase (15 U/ml) activation by this method is 8-fold less efficient than transient acidification to pH 2.5, when the LTGF-/31 concentration is 0.4 nM. At higher concentrations of L-TGF-/31 (4 nM) sialidase treatment (15 U/ml) is only 10% less effective than transient acidification, as measured by inhibition of [3H]thymidine incorporation. 38
Monosaccharides Carbohydrate structures present in the precursor remnant of L-TGF/31 (see below) appear to be important in maintaining latency. 38 Several small monosaccharides have been tested to see if they can compete with these structures. In the presence of 15 mM sialic acid or 50 mM mannose 34 y . Sato and D. B. Rifkin, J. Cell Biol. 1119, 309 (1989). 35 j. H. Elder and S. Alexander, Proc. Natl. Acad. Sci. U.S.A. 79, 4540 (1982). 36 N. T. Thotakura and O. P. Bahl, this series, Vol. 138, p. 350. 37 M. Saito, K. Sugano, and Y. Nagai, J. Biol. Chem. 254, 7845 (1979). 38 K. Miyazono and C. H. Heldin, Nature (London) 338, 158 (1989).
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6-phosphate, activation of an unspecified amount of L-TGF-fll occurs in situ and leads to inhibition of [3H]thymidine incorporation (42 and 58%, respectively. 38
Heparin The sulfated mucopolysaccharide heparin has an antiproliferative action on several cell types that is distinct from its more well-known anticoagulant activity. 39-42 Recent evidence 43 suggests that the heparin-induced inhibition of smooth muscle cell proliferation in serum-containing medium could be due to heparin-mediated dissociation of TGF-/3 (the effective inhibitor) from a2-macroglobulin (a:-m). It was previously shown ~4that LTGF-fl in serum is a complex of 25-kDa TGF-/3 associated with a2-m. Responsive cells are grown for 18 hr in 10% fetal bovine serum, which is a source of L-TGF-/3 (TGF-/3 plus a2-m), and with or without 100/zg/ml heparin. These cultures are then pulsed for 2 hr with [3H]thymidine to ascertain the inhibitory effect caused by heparin activation of serum LT G F - f l . 43
Purification of Latent Transforming Growth Factor/31 from Human Blood Platelets The starting point of the purification procedure 32 is the nonadsorbed fraction from the CM-Sephadex (Pharmacia-LKB, Uppsala, Sweden) chromatography step of the platelet-derived growth factor purification. 44 Fifteen liters of this fraction is mixed with dry QAE-Sephadex (Pharmacia P-L Biochemicals), 0.7 mg/liter, A-50, and left on a shaker overnight. After allowing the gel to sediment, the supernatant is discarded and the gel poured into a column (5 x 60 cm). The column is washed with 4 liters of 10 mM phosphate buffer, pH 7.4, containing 75 mM NaCl; then, by increasing the salt gradient, L-TGF-fil is eluted between 250 and 800 mM NaCl. Precipitation of the proteins in the latter eluate is accomplished by addition of ammonium sulfate to 35% saturation (209 mg/liter). Following a 2-hr equilibration at 4 °, the precipitated proteins are recovered by centrif39 M. M. Lippman and M. B. Mathews, Fed. Proc., Fed. Am. Soc. Exp. Biol. 36, 55 (1977). 4o j. R. Guyton, R. Rosenberg, A. Clowes, and M. Karnovsky, Circ. Res. 46, 625 (1980). 41 W. E. Benitz, J. D. Lessler, J. D. Coulson, and M. Bernfield, J. Cell. Physiol. 127, 1 (1986). 42 C. F. Reilly, L. Fritze, and R. D. Rosenberg, J. Cell. Physiol. 129, 11 (1986). 43 T. A. McCaffrey, D. J. Falcone, C. F. Brayton, L. A. Agarwal, F. G. P. Welt, and B. B. Weksler, J. Cell Biol. 109, 441 (1989). 44 C.-H. Heldin, A. Johnsson, B. Ek, S. Wennergren, L. ROnnstrand, A. Hammacher, B. Faulders, ,~. Wasteson, and B. Westermark, this series, Vol. 147, p. 3.
[31]
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ugation at 2075 g for 15 min and resuspended in about 100 ml of i0 mM Tris-HCl, pH 7.4, with 150 mM NaCI. This fraction is then extensively dialyzed against the same buffer, after which an equal volume of 2 M ammonium sulfate in 10 mM Tris-HCl, pH 7.4, is added. The sample is centrifuged at 2075 g for 15 min and the supernatant layered on a 20-ml column ofoctyl-Sepharose (Pharmacia-LKB) preequilibrated with l M ammonium sulfate in 10 mM Tris-HCl, pH 7.4. After washing the column with the same buffer, L-TGF-/31-containing material is eluted with l0 mM Tris-HCI, pH 7.4. The eluate is dialyzed against l0 mM phosphate buffer, pH 6.8, containing l0 g M CaC! 2 and injected on an HPLC hydroxyapatite column (7.8 x 100 ram, Bio-Rad, Richmond, CA), equipped with a guard column (4.0 x 50 mm, Bio-Rad). The hydroxyapaprite column is equilibrated in 10 mM phosphate, pH 6.8, containing 10 ~M CaCl,, at a flow rate of 0.5 mi/min. Fractions rich in L-TGF-/31, which elutes at an elution volume of around 20 ml, are pooled and concentrated to 100/.d with a Centricon microconcentrator (Amicon Corp., Danvers, MA). The concentrate is injected onto a Superose 6 column (HR10/30, Pharmacia-LKB), equilibrated with 500 mM NaCI, 10 mM Tris-HCI, pH 7.4, and eluted with the same buffer at a flow rate of 0.5 ml/min. L-TGF/31 elutes between thyroglobulin and ferritin at an elution volume around 14 ml; fractions containing L-TGF-/31 are pooled, mixed with an equal volume of 2.8 M ammonium sulfate (HPLC-grade, Bio-Rad), I00 mM phosphate, pH 6.8, and injected onto an alkyI-Sepharose HR 5/5 column (Pharmacia-LKB) equilibrated with 1.4 M ammonium sulfate, 100 mM phosphate, pH 6.8. The column is eluted with a gradient of 1.4-0 M ammonium sulfate in 100 mM phosphate, pH 6.8, at a flow rate of 0.5 ml/ min. L-TGF-/31 elutes from the last column between 16 and 19 ml elution volume. As performed by Miyazono et al., 3z this protocol gave a 2000fold purification. Starting with 800-1000 liters of human blood the initial nonadsorbed fraction contained 300 g of platelet protein at the beginning, and after the final step 40 p~gprotein remained. In [3H]thymidine incorporation assays the latter material gave maximal inhibition at 5 ng/ml. C o m m e n t s . Analysis by chromatography and electrophoretic methods 32 and Western blotting 3t revealed that L-TGF-/31 from human platelets is composed of an N-terminal precursor remnant present as a disulfidebonded dimer bound with disulfide bonds to an approximately 125 kDa binding protein, with the activatable TGF-/31 dimer (25 kDa) being noncovalently attached to the precursor remnant. Only the N-terminal precursor dimer remnant appears to be essential for conferring latency. 4-~The latter 45 L. E. Gentry, N. R. Webb, G. J. Lim, A. M. Brunner, J. E. Ranchalis, D. R. Twardzik, M. N. Lioubin, H. Marquardt, and A. F. Purchio, Mol. Cell. Biol. 7, 3418 (1987).
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has been shown to contain mannose 6-phosphate residues, 46 which no doubt explains why L-TGF-/31 can bind to the cation-independent mannose 6-phosphate/insulin-like growth factor II (IGF-II) receptor. 47 Platelet L-TGF-/31 appears to be structurally similar, if not identical, to the latent form secreted by other cells. 3~ H o w e v e r , one should note that this latent form is different from that in serum, which is a complex of 25-kDa TGF-fll associated with a2-m. 24 Final Remarks In the event that the other isoforms o f TGF-/3 (i.e.,/32,/33, and/34) exist as latent forms, they could well be different, not only from each other, but also from L-TGF-/31, given the dissimilarities in their respective precursor sequences. 7 It is possible that, for certain bioassays, activation o f L-TGF-/31 occurs in situ, that is, would not require prior, exogenous activation. Indeed, the usual bioassays like soft agar colony formation or inhibition o f [3H]thymidine incorporation are clearly artificial laboratory expedients. It is equally clear that, in vivo, the organism must have acquired one or more ways to activate the latent form, since the latter does not bind to TGF-/3 receptors on the surface membrane, 2~ though it can bind to the mannose 6-phosphate/IGF-II receptor ~7 (the physiological significance of the latter binding is not yet known, and most of these receptors are intracellular). The possible activation of L-TGF-/31 in acidic cellular microenvironments (tumoral masses, osteoclasts), perhaps in cooperation with other activating mechanisms, has been proposed, 48 It could be that activating treatments of purified L-TGF-/31 do not fully reflect those occurring in vivo, where the complex is present with many other factors which might modulate natural, activating processes.
Acknowledgments I thank the INSERM for financial support (Contract 884015), Drs. Thrrrse Heyman and Philippe Vigier for helpful comments on the manuscript, and Fran~oise Arnouilh for typographical assistance.
A. F. Purchio, J. A. Cooper, A. M. Brunner, M. N. Lioubin, L. E. Gentry, K. S. Kovacina, R. A. Roth, and H. Marquardt, J. Biol. Chem. 2639 14211 (1988). 47K. S. Kovacina, G. Steele-Perkins, A. F. Purchio, M. N. Lioubin, K. Miyazono, C. H. Heldin, and R. A. Roth, Biochem. Biophys. Res. Commun. 160, 393 (1989). 48p. Jullien, T. M. Berg, and D. A. Lawrence, Int. J. Cancer43, 886 (1989).
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production of chrondrocyte-specific proteoglycan with EDs0 values of approximately 0.5-1 ng/ml in the DHCB assay.
Acknowledgments We thank Dr. Hans Marquardt for sequencing bovine TGF-/32, David Schmidt for purification and characterization of TGF-/31 and TGF-/32 from bovine bones, and Andrea Thompson for comments on the description of the chondrogenesis assays.
[31] Identification and Activation of Latent Transforming Growth Factor [3
By DAVID A. LAWRENCE Introduction Transforming growth factor/3 (TGF-/3) has been the subject of several recent reviews, 1.2so here only some of its salient properties will be recalled as an anchor point for this chapter, which is specifically concerned with latent TGF-/3 (L-TGF-/3). In its active form TGF-/3 is multifunctional and can stimulate or inhibit cell proliferation, cell differentiation, and cellular function depending on cell type (e.g., fibroblast or epithelial cell) and on what other growth factors are present in the local environment. Most of our knowledge about the biological effects of TGF-/3 concern TGF-/31, simply because the other isoforms TGF-/32, -/33, and -/34 were discovered more recently. 3-7 TGF-/31 is produced by most cell types s and, unlike 1 M. B. Sporn, A. B. Roberts, L. M. Wakefield, and B. de Crombrugge, J. Cell Biol. 105, 1039 (1987). 2 D. A. Lawrence, "Malignant Cell Secretion" (V. Krsmanovic, ed.), p. 215. CRC Press, Boca Raton, Florida, 1990. 3 S. Cheifetz, J. A. Weatherbee, M. L. S. Tsang, J. K. Anderson, J. E. Mole, R. Lucas, and J. Massagu6, Cell (Cambridge, Mass.) 48, 409 (1987). 4 T. lkeda, M. N. Liobin, and H. Marquardt, Biochemistry 26, 2406 (1987). 5 p. Ten Dijke, P. Hansen, K. K. lwata, C. Pieler, and J. G. Foulkes, Proe. Natl. Acad. Sci. U.S.A. 85, 4715 (1988). 6 S. Jakowlew, P. J. Dillard, P. Kondaiah, M. B. Sporn, and A. B. Roberts, Mol. Endocrinol. 28, 747 (1988). 7 R. Derynck, P. B. Lindquist, A. Lee, D. Wen, J. Tamm, J. L. Graycar, L. Rhee, A. J. Mason, D. A. Miller, R. J. Coffey, H. L. Moses, and E. Y. Chen, EMBO J. 7, 3737 (1988). 8 R. Derynck, J. A. Jarrett, E. Y. Chert, D. H. Eaton, J. R. Bell, R. K. Assoian, A. B. Roberts, M. B. Sporn, and D. V. Goeddel, Nature (London) 316, 701 (1985).
METHODS IN ENZYMOLOGY, VOL. 198
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classic hormones, acts locally via paracrine and autocrine modes.9, ~0However, before such local action can occur, TGF-/31 must first be activated, as most of this growth regulatory molecule is released from producer cells in an inactive, latent form. ~1-14 Because TGF-fi is resistant to acid pH (pH 2.0), 15'16 purification schemes developed for TGF-/3 made full use of acetic acid for dialysis of cell-conditioned media and cell extracts and as eluant for gel-filtration columns. 17 Purification under acid conditions always yielded the biologically active 25-kDa dimer, by dissociation of the latent complex (see later). Thus, the latency phenomenon had been completely overlooked until this laboratory reported that normal chicken, mouse, and human embryo fibroblasts all released a TGF-/3 activity in a latent form under neutral conditions. I~ Quite soon after we showed that other fibroblasts, normal and transformed by various RNA and DNA viruses, also secreted L-TGF/3.12"~8Perhaps more important was the demonstration ~4that human blood platelets (with bone one of the richest sources of TGF-/319) contained a latent form of TGF-/3. Gel-filtration runs performed during the initial studies indicated that L-TGF-/3 was of high molecular weight (>400K).11'13'14 Considering the activation treatments (see later) it was proposed that LTGF-/3 was a complex of 25-kDa TGF-fl associated noncovalently with a carrier protein. ~3 Moreover, it was suggested that activation of L-TGF-/3 could be a critical step in the regulation of biological activity. 14 As a general phenomenon, the latency of TGF-/31 has been confirmed by several laboratories. 20,21 9 M. B. Sporn, A. B. Roberts, L. M. Wakefield, and R. K. Assoian, Science 233, 532 (1986). J0 A. S. Goustin, E. B. Leof, G. D. Shipley, and H. L. Moses, CancerRes. 46, 1015 (1986). tl D. A. Lawrence, R. Pircher, C. Kryc~ve-Martinerie, and P. Jullien, J. Cell. Physiol. 121, 184 (1984). J2 R. Pitcher, D. A. Lawrence, and P. Jullien, Cancer Res. 44, 5538 (1984). 13 D. A. Lawrence, R. Pitcher, and P. Jullien, Biochem. Biophys. Res. Commun. 133, 1026 (1985). 14 R. Pitcher, P. Jullien, and D. A. Lawrence, Biochem. Biophys. Res. Commun. 136, 30 (1986). 15 H. L. Moses, E. L. Branum, J. A. Proper, and R. A. Robinson, Cancer Res. 41, 2842 (1981). 16 A. B. Roberts, M. A. Anzano, L. C. Lamb, J. M. Smith, and M. B. Sporn, Proc. Natl. Acad. Sci, U.S.A. 78, 5339 (1981). 17 M. B. Sporn, M. A. Anzano, R. K. Assoian, J. E. De Larco, C. A. Frolik, C. A. Meyers, and A. B. Roberts, Cancer Cells 1, l (1984). 18 C. Kryc~ve-Martinerie, D. A. Lawrence, J. Crochet, P. Jullien, and P. Vigier, Int. J. Cancer 35, 553 (1985). t9 j. L. Carrington, A. B. Roberts, K. C. Flanders, N. S. Roche, and A. H. Reddi, J. Cell Biol. 107,1969 (1988). 2o R. J. Coffey, G. D. Shipley, and H. L. Moses, Cancer Res. 46, 1164 (1986). 2J L. M. Wakefield, D. M. Smith, T. Masui, C. C. Harris, and M. B. Sporn, J. Cell. Biol. 105, 965 (1987).
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Sample Preparation
Latent Transforming Growth Factor fi Many cell types are still cultured in the presence of serum, despite the advancing knowledge regarding growth factor supplements 2z to minimal media containing salts, amino acids, and vitamins. In vivo, however, cells only come into contact with serum at wound sites. 22A further disadvantage of using serum in the present context is that it contains numerous growth factors in variable quantities, 22 including TGF-fl essentially in a latent form. 23"24 Where no satisfactory serum-free medium has been found in which the desired cells grow, one can often avoid the problem by first growing the cells in medium with serum and then washing the cells, at least twice, with serum-free medium and reincubating them for 12 to 48 hr in the serum-free medium, which is then harvested to provide a virtually serum-free cell-conditioned medium. During the washing procedure it is important to incline the culture dish and drain it to remove as much of the initial serum-containing growth medium as possible. Some investigators reincubate the washed cultures with serum-free medium for 6-24 hr and then discard the latter before further incubation in serum-free medium, which will constitute the sample for testing. 2°'2~ This additional step removes further serum components. Clearly, each investigator must determine the culture requirements of cells used, but where serum must be used this does not necessarily preclude identifying TGF-fl in such samples. Many growth factors, including TGF-fl, can be expected to accumulate in culture medium with time; harvesting after a 24-hr period is common, and, where cell viability is maintained, repeated harvests over 4-5 days can be made. It is possible to identify activatable TGF-fl from only 1.0 ml of a crude, unconcentrated sample, but using 5.0 ml or more is simpler at the bench level. After harvesting, the sample should be centrifuged (10 min, 4000 g) to remove whole cells and most debris. Depending on the volume available, the clarified conditioned medium can be divided into 2.0- to 5.0-ml aliquots, some of which can be stocked at - 2 0 ° or below for later use. One should avoid defrosting a sample more than once, as it has been reported that repeated freezing and thawing tend to activate L-TGF-fi. 25In contrast, purified TGF-fl (25 kDa) tends to be inactivated by this treatment.
z2 D. Barnes and G. Sato, Cell (Cambridge, Mass.) 22, 649 (1980). 2s C. Bjornson-Childs, J. A. Proper, R. F. Tucker, and H. L. Moses, Proc. Natl. Acad. Sci. U.S.A. 79, 5312 (1982). 24 M. O'Connor-McCourt and L. M. Wakefield, J. Biol. Chem. 262, 14090 (1987). _~5R. M. Lyons, J. Keski-Oja, and H. L. Moses, J. Cell Biol. 106, 1659 (1988).
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Latent Transforming Growth Factor fl Neutral, nondissociating conditions must be used to prepare samples, otherwise some activation will occur. Sonication and/or homogenization (e.g., with a Dounce-type homogenizer) are convenient for preparing extracts of most cells. We have used sonication (30-sec duration at 8/zm peak to peak with a 1.0 cm diameter probe) to prepare L-TGF-fl from human blood platelets, j4 Washed platelets are taken up in phosphatebuffered saline (PBS), or other neutral buffer (1 mg wet weight/20 mi). After sonication the homogenate is centrifuged (100,000 g for 1 hr) and the resulting supernatant taken as the neutral extract. For tissue extraction the reader is referred to standard procedures for tissue maceration, bearing in mind the necessity of using neutral buffers without detergents.
Activation of Latent Transforming Growth Factor fil The most frequently used activation method is acidification, usually followed by reneutralization to render the sample biocompatible again.mJ It is convenient to prepare the following solutions: 1.0, 0.5, and 0.1 M HCI and NaOH in culture-grade, distilled water. Depending on the sample volume and the buffering capacity of its contents (e.g., serum or HEPES buffer will provide extra buffering strength), HCI is added dropwise with rapid mixing until pH 2.0 is obtained. Approximately 100/zl of 1.0 M HCI added to 2.0 ml Dulbecco's medium leads to pH 2.0. At this low pH all L-TGF-fll is activated (e.g., a 20-fold increase in activity over an original sample of conditioned medium 13 and a 300-fold increase for an acidified human platelet extract compared to the neutral extractl4), but considerable activation occurs (-7-fold) when chicken embryo fibroblast-conditioned medium, which contains latent TGF-fl, is brought to pH 5.0.13 To avoid excessive dilution during acidification, sample volumes greater than 10.0 ml should be treated with concentrated HCI (-10.0 M). The acidified sample is left for 1 hr at 4 °. (These were the original conditions for standardizing the procedure. Because activation appears to be due to pH shock dissociating the latent complex, the above time might well be shortened to about 10 min as long as this practice is followed throughout.) Reneutralization of the acidified sample is carried out by the dropwise addition of NaOH to return to the initial pH. Because pH can rise above pH 8.0, care must be exercised and use made of lower NaOH molarities to obtain the final pH. The alkaline overshoots are probably of little consequence as alkalinization is another activation treatment for L-TGF-fl. 13
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Comments. The protein concentration of serum-free conditioned medium can be quite variable depending on cell type and culture conditions, but it rarely exceeds 100 /xg/ml. Acidification of such media does not normally lead to visible turbidity unless serum is present. Such precipitated material can distort assays of activated samples and should be removed by centrifugation (10 min, 4000 g). Some microbial contamination of samples undergoing activation will inevitably occur during pH meter manipulations, but this can be cut to a minimum by using sterile pipettes, micropipette cones, and solutions of acid and base. To avoid potential losses of protein by filtration (e.g., 0.45-/xm cartridges) one may sterilize the samples for bioassay by 6°Co-irradiation, although such facilities are not generally available, leaving filtration as the more common method for sample asepsis. With an activated and sterile sample in hand, one can proceed to the actual assay of TGF-/3. The investigator can choose one or more of these standard assays: stimulation of colony formation of NRK-49F fibroblasts in soft agar in the presence of epidermal growth factor (EGF)26;inhibition of [3H]thymidine incorporation in certain sensitive cell lines, like the mink epithelial cell line CCL6427;radioreceptor competition assay28'29; and neutralization or Western blotting with specific anti-TGF-/3 antiserum. 3°,3j The first three assays can be used with appropriate standards of known TGF-/3 concentrations to determine less than 1.0 ng/ml TGF- 3. As a control for activated samples of TGF-/3 one should check, in addition to the original sample, an aliquot which received premixed amounts of acid and base (this sample ought to give the same result as the original unactivated sample). For assays on conditioned media, one should test an aliquot of culture medium that has not been in contact with the cells. One-half of the aliquot should be tested directly and the other half should receive the acid/base treatment. Secretion of spontaneously active L-TGF-/31 appears to be relatively uncommon. For large samples needing further purification, another convenient acid-activating treatment, is dialysis in acetic acid. Both I% and I M 26 j. E. De Larco and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 75, 4001 (1978). 27 R. F. Tucker, G. D. Sfiipley, H. L. Moses, and R. W. Holley, Science 226, 705 (1984). 2~ C. A. Frolik, L. M. Wakefield, D. M. Smith, and M. B. Sporn, J. Biol. Chem. 259, 10995 (1984). 29 R. F. Tucker, E. L. Branum, G. D. Shipley, R. J. Ryan, and H. L. Moses, Proc. Natl. Acad. Sci. U.S.A. 81, 6757 (1984). 30 T. Masui, L. M. Wakefield, J. F. Lechner, M. A. LaVeck, M. B. Sporn, and C. C. Harris, Proc. Natl. Acad. Sci. U.S.A. 83, 2438 (1986). st L. M. Wakefield, D. M. Smith, K. C. Flanders, and M. B. Sporn, J. Biol. Chem. 263, 7646 (1988).
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(5.7%) acetic acid solutions can be employed. Acetic acid (1 M) gives a pH of 2.4, low enough to activate all L-TGF-/3. As acetic acid is evaporated during lyophilization, samples thus treated can be reconstituted in the desired buffer or culture medium and also concentrated at the same time by appropriate volume reduction. Other Methods of Activating Latent Transforming Growth Factor/31
Boiling Boiling is a convenient procedure for 20 or more samples (e.g., column fractions or conditioned media from several cell types or from various culture conditions) where acidification/neutralization becomes tedious. The sample is kept in boiling water for 3 min. This heat treatment is about 50% less effective than acidification. ~3Human platelet L-TGF-/31 cannot be activated by boiling, 3z whereas that of rat platelets can be activated in this way. 33
Alkalinization Alkalinization is simply the reverse of the acidification procedure. The pH of the sample is brought to pH 11.0 or more by dropwise addition of NaOH before reneutralization with HCI.
Urea Exposure of the sample to 8 M urea at room temperature activates LTGF-/31 but tends to be a less effective method for L-TGF-/31 in conditioned media 13 and platelet extracts.14
Enzymatic Activation By Plasmin. A 2-hr incubation at 37° with 0.2 U plasmin per milliliter conditioned medium gives slightly less than one-half the activation obtained by acidifying to pH 1.5 (respectively 450 and 1000 colonies in the soft agar assay).25 The plasmin treatment is stopped by adding phenylmethylsulfonyl fluoride and aprotonin at 2.0 mM and 0.55 TIU/ml final concentrations followed by 1.0 mg bovine serum albumin (as a carrier) per milliliter of reaction mixture. In the TGF-/3 radioreceptor assay, maximum inhibition is obtained following treatment with 0.5 U/ml plasmin. Evidence 32 K. Miyazano, U. Hellman, C. Wernstedt, and C. H. Heldin, J. Biol. Chem. 263, 6407 (1988). 33 T. N a k a m u r a , T. Kitazawa, and A. lchihara, Biochem. Biophys. Res. Commun. 141, 176 (1986).
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is provided 25 for the existence of two pools of L-TGF-/3, one being activated by plasmin (or mild acid pH 4.5) and the other by strong acid at pH 1.5. Further evidence for plasmin activation comes from coculture experiments. 34 Here, the migration of confluent bovine aortic endothelial cells into a "wounded area" (obtained by scraping across an adherent cell monolayer in a petri dish with a razor blade) is blocked by cocultivating bovine pericytes or smooth muscle cells in the denuded area. This blockage appears to be due to plasmin-mediated activation of L-TGF-/3 in situ. By Endoglycosidase F. Endoglycosidase F (endo F) removes complex and oligomannose (high mannose) N-linked carbohydrates. 35-37L-TGF-/31 purified from human platelets (see below) is incubated at 37 ° for 16 hr in 50.0 mM EDTA and 100.0 mM sodium phosphate, pH 7.4, containing 10 or 25 U of endo F from Flavobacterium meningosepticum (Boehringer, Mannheim, Germany) per milliliter of reaction mixture. 38 In a radioreceptor assay, 0.5 nM (purified) L-TGF-/31 pretreated with 10 or 25 U/ml of endo F gives the same degree of inhibition as does 0.1 nM L-TGF-/31 transiently acidified to pH 2.0. By Sialidase (N-Acetylneuraminidase). Several enzymes have the general name sialidase; depending on their specificity, they break particular linkages to remove N-acetylneuraminic acid from various polysaccharides, glycoproteins, and gangliosides. 36.37The sialidase used in the report 38 cited here is type V from CIostridium perfringens (Sigma, St. Louis, MO). Purified L-TGF-/31 at 320 pM is incubated with 2 U of sialidase per milliliter of reaction mixture in 100 mM sodium phosphate buffer, pH 5.6, for 18 hr at 37°. The effect of the treatment is assayed by the inhibition of [3H]thymidine incorporation; the extent of inhibition is about 80%. Even with saturating concentrations of sialidase (15 U/ml) activation by this method is 8-fold less efficient than transient acidification to pH 2.5, when the LTGF-/31 concentration is 0.4 nM. At higher concentrations of L-TGF-/31 (4 nM) sialidase treatment (15 U/ml) is only 10% less effective than transient acidification, as measured by inhibition of [3H]thymidine incorporation. 38
Monosaccharides Carbohydrate structures present in the precursor remnant of L-TGF/31 (see below) appear to be important in maintaining latency. 38 Several small monosaccharides have been tested to see if they can compete with these structures. In the presence of 15 mM sialic acid or 50 mM mannose 34 y . Sato and D. B. Rifkin, J. Cell Biol. 1119, 309 (1989). 35 j. H. Elder and S. Alexander, Proc. Natl. Acad. Sci. U.S.A. 79, 4540 (1982). 36 N. T. Thotakura and O. P. Bahl, this series, Vol. 138, p. 350. 37 M. Saito, K. Sugano, and Y. Nagai, J. Biol. Chem. 254, 7845 (1979). 38 K. Miyazono and C. H. Heldin, Nature (London) 338, 158 (1989).
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6-phosphate, activation of an unspecified amount of L-TGF-fll occurs in situ and leads to inhibition of [3H]thymidine incorporation (42 and 58%, respectively. 38
Heparin The sulfated mucopolysaccharide heparin has an antiproliferative action on several cell types that is distinct from its more well-known anticoagulant activity. 39-42 Recent evidence 43 suggests that the heparin-induced inhibition of smooth muscle cell proliferation in serum-containing medium could be due to heparin-mediated dissociation of TGF-/3 (the effective inhibitor) from a2-macroglobulin (a:-m). It was previously shown ~4that LTGF-fl in serum is a complex of 25-kDa TGF-/3 associated with a2-m. Responsive cells are grown for 18 hr in 10% fetal bovine serum, which is a source of L-TGF-/3 (TGF-/3 plus a2-m), and with or without 100/zg/ml heparin. These cultures are then pulsed for 2 hr with [3H]thymidine to ascertain the inhibitory effect caused by heparin activation of serum LT G F - f l . 43
Purification of Latent Transforming Growth Factor/31 from Human Blood Platelets The starting point of the purification procedure 32 is the nonadsorbed fraction from the CM-Sephadex (Pharmacia-LKB, Uppsala, Sweden) chromatography step of the platelet-derived growth factor purification. 44 Fifteen liters of this fraction is mixed with dry QAE-Sephadex (Pharmacia P-L Biochemicals), 0.7 mg/liter, A-50, and left on a shaker overnight. After allowing the gel to sediment, the supernatant is discarded and the gel poured into a column (5 x 60 cm). The column is washed with 4 liters of 10 mM phosphate buffer, pH 7.4, containing 75 mM NaCl; then, by increasing the salt gradient, L-TGF-fil is eluted between 250 and 800 mM NaCl. Precipitation of the proteins in the latter eluate is accomplished by addition of ammonium sulfate to 35% saturation (209 mg/liter). Following a 2-hr equilibration at 4 °, the precipitated proteins are recovered by centrif39 M. M. Lippman and M. B. Mathews, Fed. Proc., Fed. Am. Soc. Exp. Biol. 36, 55 (1977). 4o j. R. Guyton, R. Rosenberg, A. Clowes, and M. Karnovsky, Circ. Res. 46, 625 (1980). 41 W. E. Benitz, J. D. Lessler, J. D. Coulson, and M. Bernfield, J. Cell. Physiol. 127, 1 (1986). 42 C. F. Reilly, L. Fritze, and R. D. Rosenberg, J. Cell. Physiol. 129, 11 (1986). 43 T. A. McCaffrey, D. J. Falcone, C. F. Brayton, L. A. Agarwal, F. G. P. Welt, and B. B. Weksler, J. Cell Biol. 109, 441 (1989). 44 C.-H. Heldin, A. Johnsson, B. Ek, S. Wennergren, L. ROnnstrand, A. Hammacher, B. Faulders, ,~. Wasteson, and B. Westermark, this series, Vol. 147, p. 3.
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TGF-/3
335
ugation at 2075 g for 15 min and resuspended in about 100 ml of i0 mM Tris-HCl, pH 7.4, with 150 mM NaCI. This fraction is then extensively dialyzed against the same buffer, after which an equal volume of 2 M ammonium sulfate in 10 mM Tris-HCl, pH 7.4, is added. The sample is centrifuged at 2075 g for 15 min and the supernatant layered on a 20-ml column ofoctyl-Sepharose (Pharmacia-LKB) preequilibrated with l M ammonium sulfate in 10 mM Tris-HCl, pH 7.4. After washing the column with the same buffer, L-TGF-/31-containing material is eluted with l0 mM Tris-HCI, pH 7.4. The eluate is dialyzed against l0 mM phosphate buffer, pH 6.8, containing l0 g M CaC! 2 and injected on an HPLC hydroxyapatite column (7.8 x 100 ram, Bio-Rad, Richmond, CA), equipped with a guard column (4.0 x 50 mm, Bio-Rad). The hydroxyapaprite column is equilibrated in 10 mM phosphate, pH 6.8, containing 10 ~M CaCl,, at a flow rate of 0.5 mi/min. Fractions rich in L-TGF-/31, which elutes at an elution volume of around 20 ml, are pooled and concentrated to 100/.d with a Centricon microconcentrator (Amicon Corp., Danvers, MA). The concentrate is injected onto a Superose 6 column (HR10/30, Pharmacia-LKB), equilibrated with 500 mM NaCI, 10 mM Tris-HCI, pH 7.4, and eluted with the same buffer at a flow rate of 0.5 ml/min. L-TGF/31 elutes between thyroglobulin and ferritin at an elution volume around 14 ml; fractions containing L-TGF-/31 are pooled, mixed with an equal volume of 2.8 M ammonium sulfate (HPLC-grade, Bio-Rad), I00 mM phosphate, pH 6.8, and injected onto an alkyI-Sepharose HR 5/5 column (Pharmacia-LKB) equilibrated with 1.4 M ammonium sulfate, 100 mM phosphate, pH 6.8. The column is eluted with a gradient of 1.4-0 M ammonium sulfate in 100 mM phosphate, pH 6.8, at a flow rate of 0.5 ml/ min. L-TGF-/31 elutes from the last column between 16 and 19 ml elution volume. As performed by Miyazono et al., 3z this protocol gave a 2000fold purification. Starting with 800-1000 liters of human blood the initial nonadsorbed fraction contained 300 g of platelet protein at the beginning, and after the final step 40 p~gprotein remained. In [3H]thymidine incorporation assays the latter material gave maximal inhibition at 5 ng/ml. C o m m e n t s . Analysis by chromatography and electrophoretic methods 32 and Western blotting 3t revealed that L-TGF-/31 from human platelets is composed of an N-terminal precursor remnant present as a disulfidebonded dimer bound with disulfide bonds to an approximately 125 kDa binding protein, with the activatable TGF-/31 dimer (25 kDa) being noncovalently attached to the precursor remnant. Only the N-terminal precursor dimer remnant appears to be essential for conferring latency. 4-~The latter 45 L. E. Gentry, N. R. Webb, G. J. Lim, A. M. Brunner, J. E. Ranchalis, D. R. Twardzik, M. N. Lioubin, H. Marquardt, and A. F. Purchio, Mol. Cell. Biol. 7, 3418 (1987).
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has been shown to contain mannose 6-phosphate residues, 46 which no doubt explains why L-TGF-/31 can bind to the cation-independent mannose 6-phosphate/insulin-like growth factor II (IGF-II) receptor. 47 Platelet L-TGF-/31 appears to be structurally similar, if not identical, to the latent form secreted by other cells. 3~ H o w e v e r , one should note that this latent form is different from that in serum, which is a complex of 25-kDa TGF-fll associated with a2-m. 24 Final Remarks In the event that the other isoforms o f TGF-/3 (i.e.,/32,/33, and/34) exist as latent forms, they could well be different, not only from each other, but also from L-TGF-/31, given the dissimilarities in their respective precursor sequences. 7 It is possible that, for certain bioassays, activation o f L-TGF-/31 occurs in situ, that is, would not require prior, exogenous activation. Indeed, the usual bioassays like soft agar colony formation or inhibition o f [3H]thymidine incorporation are clearly artificial laboratory expedients. It is equally clear that, in vivo, the organism must have acquired one or more ways to activate the latent form, since the latter does not bind to TGF-/3 receptors on the surface membrane, 2~ though it can bind to the mannose 6-phosphate/IGF-II receptor ~7 (the physiological significance of the latter binding is not yet known, and most of these receptors are intracellular). The possible activation of L-TGF-/31 in acidic cellular microenvironments (tumoral masses, osteoclasts), perhaps in cooperation with other activating mechanisms, has been proposed, 48 It could be that activating treatments of purified L-TGF-/31 do not fully reflect those occurring in vivo, where the complex is present with many other factors which might modulate natural, activating processes.
Acknowledgments I thank the INSERM for financial support (Contract 884015), Drs. Thrrrse Heyman and Philippe Vigier for helpful comments on the manuscript, and Fran~oise Arnouilh for typographical assistance.
A. F. Purchio, J. A. Cooper, A. M. Brunner, M. N. Lioubin, L. E. Gentry, K. S. Kovacina, R. A. Roth, and H. Marquardt, J. Biol. Chem. 2639 14211 (1988). 47K. S. Kovacina, G. Steele-Perkins, A. F. Purchio, M. N. Lioubin, K. Miyazono, C. H. Heldin, and R. A. Roth, Biochem. Biophys. Res. Commun. 160, 393 (1989). 48p. Jullien, T. M. Berg, and D. A. Lawrence, Int. J. Cancer43, 886 (1989).
