Latent Transforming Growth Factor-@ from Human Platelets

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 263, No. 16, Issue of June 5 , pp. 7646”7654,1988 Printed in U.S.A .

Latent Transforming Growth Factor-@ from Human Platelets AHIGH MOLECULAR WEIGHTCOMPLEX

CONTAINING PRECURSOR SEQUENCES* (Received for publication, September 10,1987)

Lalage M. Wakefield$, Diane M. Smith, Kathleen C. Flanders, andMichael B. Sporn From the Laboratory of Chemopreuention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Human platelets, when induced to degranulate by thrombin, secrete transforming growth factor-@ (TGF@)in a biologically latent form. In this form, TGF-@ cannot bind to its cellular receptor, nor can it be immunoprecipitated by polyclonal antiserato TGF-@, suggesting that the receptor-binding site and other TGF-@epitopes may be masked. Western blot analysis of the platelet secretate indicatesthat the latentform of TGF-@is a 220-235 kDa complex, in which mature TGF-@(25 kDa) is noncovalently associated with sequences from the remainder of the precursor(74 kDa), and a third unidentified entity (-135 kDa). The third component is immunologically unrelatedtoother growth factor binding proteins. The complex is glycosylated, andgel filtration analysissuggests it may exist in solution as higher molecular weight aggregates. Further chromatographic analysis indicatesthat in its latent form, the platelet TGF-@cannot bind to az-macroglobulin (aZM),but that if the platelet latent TGF-@ is activated by transient acidification, the released active TGF-@will bind to azM. We have previously identified the latent formof TGF-@found in serum as an a2M*TGF-@ complex(O’Connor-McCourt, M.D., and Wakefield, L. M. (1987) J. Biol. Chen. 262, 14090-14099). We now propose that the latentTGF/3 secreted by platelets may be a cellular deliverycomplex, whereas the latent form found in serum may represent a clearance complex. Thus azM may scavenge excess TGF-@that is released when the platelet latent form is activated, possibly by the clotting process. Finally, we have shown that the latent form of TGF-@secreted by a variety of cell types in cultureis similar, if not identical tothat secreted by platelets.

Although TGF-/3 wasoriginally characterized by its ability to induce the phenotypic transformation of normal indicator cells (21, subsequent work showing an almost universal distribution of both TGF-8 and its receptor in normal tissues has indicated that TGF-@is a critical regulatory molecule in many physiological processes. It has potent proliferative and anti-proliferative effects on normal cell types and modulates expression of cell function in awide variety of in vitrosystems (for reviews, see Refs. 1 and 3). Since TGF-B often appears to act by antagonizing or modifying the action of other growth factors, hierarachically it may play an organizing role in directing growth factor action. TGF-@is secreted by cells in culture in a biologically latent form (4-6). This can be activated in vitro by transient acidification, alkalinization, or action of chaotropic agents (7), suggestingthe latentform may involvea noncovalent complex of active TGF-@with a component that confers biological latency. The secretion of TGF-@ in a latentform is conceptually important since it potentially limitsthe cellular targets of TGF-@action to those cells that areeither able themselves to activate the latent form or that are in close proximity to others that can do so. Cells that lose the ability to activate TGF-@might no longer be subject to autocrine or paracrine regulation by the molecule. This is of particular interest in view of increasing evidence suggesting that TGF-/3 may bean endogenous autocrine inhibitor of epithelial cell growth (810). In this case loss of ability to activate autocrine latent TGF-P could result in uncontrolled proliferation of these cells and contribute to malignant transformation (11, 12). Thus, the identification of the nature of the latent form and the physiological mechanism of its activation are of paramount importance in (a) understanding the control of TGF-@ action, ( b ) determining the target tissues for the molecule, and (c) establishing whether cell growth or function might be regulated in an autocrine manner by TGF-@in vivo. Transforming growth factor-@ (TGF-8)’is the parent molThe highest concentrations of TGF-P in thebody are found ecule of a growing family of structurally relatedpeptides that in the platelets, probably reflecting a major in vivo role for are involved in the regulation of cell growth and function, TGF-@ in wound healing (13). TGF-P was first purified to particularly during development and repair of tissues (1). homogeneity and characterized from this source (14). The active molecule is a 25-kDa disulfide-linked homodimer, and * The costs of publication of this article were defrayed in part by the cDNA sequence predicts that themonomer is encoded as the payment of page charges. This article must therefore be hereby the COOH-terminal 112 amino acids of a 391-amino acid marked “aduertisement” in accordance with 18 U.S.C. Section 1734 precursor (15). Pircher et al. (16) have shown that TGF-P is solely to indicate this fact. A preliminary report of these data was presented at the UCLA stored in human blood platelets as a high molecular weight Symposium on Growth Regulation of Cancer a t Park City, Utah in complex that is biologically latent andcan be activated in the same way as the latentform secreted by cells in culture. The January 1987. $ T o whom correspondence should be addressed Bldg. 41, Rm. present studieswere designed to determine whether the latent B1111,National Cancer Institute, Bethesda, MD 20892. TGF-P is activated prior to secretion when plateletsare The abbreviations used are: TGF-8, transforming growth factor- induced to degranulate and to characterize the biochemical p; aZM,a2-macroglobulin;BS3, bis-sulfosuccinimidyl suberate; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagles medium; nature of the platelet latent TGF-P. EGF, epidermal growth factor; FPLC, fast protein liquid chromatography; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NGF, nerve growth factor; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.

EXPERIMENTALPROCEDURES

Platelet Degranulation-Fresh human platelets (American Red Cross, Washington D. C.) werepurified by differential centrifugation,

7646

Latent Transforming Growth

Factor-@from Human

resuspended to 3-4 X IO9 platelets/mlin Dulbecco's phosphatebuffered saline, and then induced to degranulate by addition of 1 unit/ml of human thrombin (Behring Diagnostics) for 10 min at 37 "C.The reaction was stopped on ice, and BSA (50 pg/ml) and the proteaseinhibitorsleupeptin (1 pg/ml), pepstatin (1 pg/ml), and aprotinin (3 pg/ml) were added. Degranulated platelet aggregates were removed by centrifugation a t 2500 X g for 1 h a t 4 "C, and the supernatant, designated "platelet secretate," was aliquotted and stored a t -20 "C. This typically contained 50-200 ng/ml of TGF-@ and -200-300 pg/ml protein, with the TGF-8 recovery being 5002000 molecules/platelet. The platelet secretate was either used without furtherconcentration or was concentrated 5-10-fold prior to use by dialysis against %O x PBS or 0.15 M ammonium acetate, followed by lyophilization and resuspension, where indicated in the text. Preparation of Cell-conditioned Media"HT1080, A549, and RD cell lines were obtained from the American Type Culture Collection (Rockville, MD). RCE cells and Ha-ras transfected Fischer rat 3T3 cells were the generous gifts of Ulf Rapp (National Cancer Institute, Frederick, MD) and David Stern (Whitehead Institute for Biomedical Research, Cambridge, MA), respectively. To prepare conditioned medium, cells weregrown to -95% confluence in T150 flasks in DMEM containing 5% fetal bovine serum. Cultures were rinsed with serum-free DMEM and then washed overnight with 25 ml/flask DMEM. This medium was discarded, and cultures were incubated with 25 ml/flask fresh DMEM for 24 h. Aprotinin (1 pglml), pepstatin (0.5 pg/ml) and leupeptin (0.5 pg/ml) were added, and theconditioned media were clarified by centrifugation at 2500 X g,, for 15 min in siliconized tubes. Clarified conditioned media were concentrated 50100-fold by ultrafiltration using a YM-30 membrane (Amicon Corp., Lexington, MA). Both theultrafiltration unit and the membrane were conditioned with a PBS, 0.1% BSA solution before use. Samples were stored at -20 "C. Chemical Cross-linking and Transient Acidification-Samples were chemically cross-linked prior to analysis by reaction with 2 mM bissulfosuccinimidyl suberate (Pierce Chemical Co.) for 20 min at 4 "C. The reaction was quenched by addition of glycine to 100 mM. For some experiments the platelet secretate was transiently acidified to pH 2.5-3.0 with 5 N HCl for 5 min a t 22 "C and then reneutralized with 5 N NaOH, 1 M Hepes before use. This procedure did not result in anyloss of protein from the samples due to acid denaturation (data not shown). Matched neutral samples had premixed HCl/NaOH/ Hepes added to give the same final ionic strength. Antibodies-Immunoglobulin fractions of a rabbit polyclonal antiserum to purified human platelet TGF-@(type 1) were prepared as described previously (17). For Western blots, the antiserum was affinity purified by passage over a column of TGF-P-Sepharose, prepared by coupling 500 pg of TGF-@1 to 0.7 g of cyanogen bromideactivated Sepharose (Pharmacia LKB Biotechnologies Inc.). Briefly, the protein pellet from a 50% ammonium sulfate cut of the whole serum was dialyzed against 0.5 M NaCl, 0.1 M sodium tetraborate, pH 8.0 (wash buffer) and was loaded onto the column in this buffer a t a flow rate of 30 ml/h. After extensive washing with wash buffer, the bound material was eluted with 6 M urea, pH adjusted to 3.5 with acetic acid, and fractions were immediately reneutralized with % vol of 3 M Tris. C1, pH 8.8. Peak fractions were then pooled and dialyzed against PBS. The final immunoglobulin concentration was typically 10-15 pg/ml. Affinity purified antiserum was stored at 4 "C with 0.02% merthiolate as a preservative. This antiserum was specific for type 1TGF-@ anddid not recognize type 2 TGF-@in either Western blots or radioimmunoassay (data not shown). Rabbit polyclonal antisera were also raised to synthetic peptides corresponding to amino acids 46-56,91-102, and 267-279of the TGF-81 precursor sequence' deduced from the human cDNA clone (15) and tosynthetic peptides corresponding to amino acids 7-19 and 96-110 of the murine EGF-binding protein sequence (18).Peptides were coupled to ovalbumin or soybean trypsininhibitorthrough terminal cysteine residues, using n-maleimidobenzoyl sulfosuccinimide (Pierce Chemical Co.), and rabbits were innoculated intrader'The numbering system for the human type 1 TGF-8 sequence used in this manuscript corresponds to thatof the original sequence published by Derynck et aL (see Ref. 15). However, these authors have subsequently amended their sequence with the deletion of residue 160 (Derynck R., Rhee, L., Chen, E. Y., and Van Tilburg, A. (1987) NucleicAcidsRes. 15, 3188-3189). Thus, according to the amended sequence, the precursor protein isonly 390 amino acids long and oursynthetic peptideP267-279 corresponds to residues 266-278. The other peptides are unaffected.

Platelets

7647

mally at multiple sites on the back with 1mg of coupled peptide for the initial immunization, followed by boosts of 0.5 mgof coupled peptide a t 3-week intervals. Rabbits were bled 7 days after boosting. Antipeptide antisera were used without further purification. Sheep and goat antisera to human urinary kallikrein were the generous gift of Dr. Jack Pierce (National Institutes of Health, Bethesda, MD). Mouse EGF-binding protein was the generous gift of Dr. Paul Isackson (University of California, Irvine, CAI. Western Blots-Platelet secretate (50 p l ) was solubilized in an equal volume of sample buffer (8 M urea, 2% SDS, 125 mM Tris, pH 6.8) by boiling for 3 min, with or without 50 mM dithiothreitol. Where indicated platelet secretate was transiently acidified and/or chemically cross-linked with BS3 (see above) prior to addition of sample buffer. Electrophoresis was performed by the method of Laemmli (19) using 3-10% linear gradient gels, unless otherwise indicated. Immunoblotting was performed essentially as described by Towbin et al. (20). Briefly, proteins were transferred to nitrocellulose paper (0.45-pmpore size: Schleicher & Schuell) or Immobilon PVDF transfer membrane (Millipore Corp., Bedford, MA) overnight at 30V, followed by 1 h at 100 V. Blots were blocked in Tris-buffered saline, pH 8.2 (TBS), containing 5% BSA for 45 min a t 37 "C. They were then probed in a dilute solution of specific antiserum in TBS, 0.1% BSA, 1%normal goat serum (or normal rabbit serum if the specific antiserum was from goat) for -4 h at 22 "C. Affinity purified antiTGF-@antiserum and the anti-precursor peptide antisera were used at a final dilution of 1:50, whereas anti-kallikrein antisera were used at 1:250. To demonstrate band specificity, identical blots were probed with specific antisera blocked with matched peptide (1 pg/ml for TGF-@,50pg/ml for synthetic peptides and kallikrein). Blots probed with rabbit or goat antisera were developed using the Immunogold procedure with silver enhancement (Janssen Life Science Products, Piscataway, NJ), incubating the blot with gold-labeled second antibody at 1:lOO dilution in TBS, 0.1% BSA, 0.4% gelatine, overnight a t 22 "C. Blots probed with sheep antisera were developed with phosphatase-labeled anti-sheep antibody (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium for color development. The affinity purified anti-TGF-0 antibodies at a dilution of1:50 could detect 1-5 ng of TGF-01, and did not detect upto 100 ng of TGF-02. The anti-kallikrein antibodies could detect 4 0 ng of human urinary kallikrein. The proteinstandards used to calibrate the gelswere myosin (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), and a-chymotrypsinogen (25.7 kDa), obtained from Bethesda Research Laboratories. On some gels, a-2macroglobulin (340 kDa) from Boehringer Mannheim was included to demonstrate nonidentity of high molecular mass bands with this marker. However, since the protein migrates anomalously on SDS gels due to its heavy glycosylation, it was not used for molecular weight determination. N-Glycannse Digestion-Platelet secretate was denatured by boiling for 5 min in the presence of 0.5% SDS and 0.1 M P-mercaptoethanol. Sodium phosphate, pH 8.6, was added to a final concentration of 0.2 M, Nonidet P-40 was added to 1.25% (v/v) and N-glycanase (Genzyme, Boston, MA)was added to 10 units/ml. Samples were digested overnight a t 37 "C. Control samples were treated identically except that the enzyme was omitted. No change in the mobility of non-glycosylated proteins such as TGF-P was observed after incubation with N-glycanase, confirming the absence of contaminating protease activity in the N-glycanase preparation (data not shown). Other Reagents and Assays-Quantitative radioreceptor assays for TGF-8, with and without transient sample acidification, and the specific enzyme-linked immunosorbent assay for human a2M were performed as previously described (21), as were the soft agar assays (2). Unless otherwise indicated, TGF-@was routinely quantitated using the radioreceptor assay. Electrophoretically homogeneousTGF81, prepared from human platelets (14) was used as astandard. Proteindetermination was by the method of Bradford (22). For determination of the lectin binding specificity of the platelet latent TGF-P, all lectin 'matrices were obtained from Vector Laboratories Inc. (Burlingame, CA), except for wheat germ agglutinin which came from Sigma. For the radioimmunoassay for TGF-@,platelet secretate (neutralortransiently acidified) was diluted inPBS, 0.1%BSA. Protein ASepharose-purified anti-TGF-@ rabbit immunoglobulin was added to a final concentration of 80 pg of immunoglobulin/ml (total assay volume of 125 pl) and incubated for 1h a t 22 'C. The antiserum is limiting at thisconcentration. T - T G F - p was then added to a final concentration of -80 p~ (-49,000 cpm/tube) and incubated for a further 2 h a t 22 "C. Finally, 1 ml/tube of a 0.25% suspension of

7648

Latent Transforming Growth Factor-@from Human Platelets

washed, formaldehyde-fixed Staphylococcus A (Boehringer Mannheim) in PBS containing 0.5 mg/ml BSA, 0.1 mg/ml azide, 0.25% Nonidet P-40, and 0.05% SDS was added. (Higher detergent concentrations appeared to activate the latent complex). After 1 h of incubation, the immunoprecipitate was collected by centrifugation and counted in a y-counter,using a TGF-@standard curve over the range 1-100 ng.

TGF-j3 antibodies reveal only a single specificband at 25 kDa, characteristic of mature TGF-j3(Fig.2A, lanes 1 and 2). However, if the platelet secretate is chemically cross-linked with BS3 prior to electrophoresis, a specific high molecular mass band of220-235 kDa is now detected in the neutral sample, and only to a much lesser extent in the sample that was activated by transient acidification prior to cross-linking RESULTS (Fig. 2A, lanes 3 and 4). Specific bands were identified by comparison with an identical blot probedwith antiserum Platelets Secrete lG'F-8 in a Biologically Latent Form blocked with 1pg/ml TGF-j3 (Fig. 2A, lanes 5-8). The results The TGF-j3 secreted by thrombin-treated human platelets suggest that platelet latent TGF-j3 may be a high molecular is 290% biologically inactive as judged by its inability to weight noncovalent complex that is dissociated on exposure promote extensive colony formation by normal rat kidney to electrophoresis buffer or low pH. Dissociation by acidififibroblasts (NRK 49F) in soft agar, without prior acidification cation appears to be irreversible under the experimental conof the secreted material in vitro (Fig. 1A). Similarly >99% of ditions used (25 min of incubation at 4 "C after reneutralizathe secreted TGF-8 is unable to bind to the specific TGF-j3 tion). Scanning densitometry indicates that the220-235-kDa receptor on A549 human lung carcinoma cells without prior band represents -50% of the total detectable TGF-8. Howacidification (Fig. lB),suggesting that platelet TGF-j3 may ever, since one cannot assume that the immunoreactive epibe biologically latent because of its inability to bind to the topes on the TGF-j3 moleculeare equally accessibleor reactive receptor. Essentially identical results are obtained with the after treatment with acid or BS3, or when associated with NRK 49F receptor (data not shown). Finally, a polyclonal other proteins, the true distribution of TGF-8 between the rabbit antiserum to TGF-j3 which has been shown to inhibit different molecular weight speciescannot be quantitated acthe binding of TGF-8 to its receptor (17), cannot recognize curately from Western blots. Increasing the concentration of the platelet latent TGF-j3 unless this is first acidified (Fig. BS3, in an attempt to increase the cross-linking efficiency, 1C). These data allsuggest that thereceptor-binding site and results in a loss of immunoreactivity from both 25 kDa and probably otherTGF-8 epitopes are masked in the latent 220-235-kDa bands (data not shown). complex. Antibodies to synthetic peptides corresponding to amino Control experiments demonstrated that thrombin itself will acids 46-56,91-102 (data not shown), and 267-279of the not activate the latent form ofTGF-j3, nor will it degrade putative TGF-j3 precursor sequence also detect the same high active TGF-j3 (data notshown). The same proportion of active molecular mass band (220-235 kDa) in neutral cross-linked to latent TGF-8 was obtained when bovine type I collagen samples (Fig. 3A, lanes 1 and 5). Thus, the latent TGF-8 (12 pg/ml: Collaborative Research Inc.,Bedford,MA)was complex appears to include mature TGF-8 noncovalently used as a secretagoguein place of thrombin, although collagen associated with the precursor sequences that remain after was only able to induce the secretion of half as much total TGF-j3 as was released by thrombin (data not shown). Thus, A B it is unlikely that thethrombin is inducing artifacts as result a ANTI-TGFP of any proteolytic activity on the secreted material. I I Western Blots of Latent TGF-j3 Nonreducing Conditions-Western blots of both neutral and transiently acidified platelet secretate probed with anti-

I

-,.,

BS3

--- --+

+

+ + + + - - + +

N A N A N A N A

- -.

...,.d1

FIG. 1. Biological latency of TGF-B secreted by platelets. Neutral or transiently acidified platelet secretate ( P S ) were tested for TGF-8 activity in a variety of assays. A , colony formation by NRK 49F cells. NRK 49F cells were assayed for their ability to form colonies in soft agar in response to PS, in the presence of 10% calf serum and 1 nM EGF, as described (2). B, binding to the TGF-@ receptor on A549 cells. The ability of PS to compete with '*'I-TGF-@ for binding to the cellular receptor was determined using a two-step quantitative radioreceptor assay for TGF-@,as described (21). This format of assay prevents any excess of binding protein scoring as a false positive for [email protected] control samples had 6,960 90 cpm bound, and nonspecific binding, determined in the presence of 3 nM unlabeled TGF-@,was 1,602 k 66 cpm. C, immunoprecipitation by polyclonal antisera to [email protected] ability of PS tocompete with '*'ITGF-@ for binding to anti-TGF-@antibodies was determined as described under "Experimental Procedures." Control binding was 33,028 f 645 cpm. Results in A and C are the mean & standard deviations of three determinations, while those in B are themean of two determinations that differed by 670 kDa, partially overlapping the a2M peak (Fig. 6). A two-stepformat for the TGF-/3 radioreceptor assay was used for the column fractions. Cells were sequentially exposed to sample and then, afterwashing, to ‘“I-TGF-/3 (21) so that only TGF-/3and no binding proteins

ELUTION VOLUME Imll

FIG. 7. Anion exchange chromatography of platelet secretate. Platelet secretate was extensively dialyzed against 20 mM TrisHCl, pH 7.5, and 0.5 ml (126 ng of TGF-P, 72pg of protein) was loaded onto a Mono Q HR5/5 FPLC anion exchange column. The column was eluted with a 0-500 mM linear gradient ofNaC1, at a pressure of 0.8 mega Pascal, over a volume interval of30 ml. The flow rate was 0.5 ml/min, and 1-ml fractions were collected, with BSA and aprotinin added as in Fig. 6. Fractions were extensively dialyzed against PBS in a microdialysis apparatus using Spectrapor 3 dialysis membrane with a 3500 M , cutoff, prior to assay for apM and TGF-fl as described under “Experimental Procedures.” Both gel filtration and ion-exchange columns were preconditioned by chromatography of 10 mg of BSA under identical conditions to the test runs prior to use.

Latent Transforming Growth Factor-@from Human Platelets

7652

different properties from the platelet-derived material. Fig. 8 shows the results of Mono Q anion exchange analysis of complex formation between pure serum azM andeither highly purified active platelet TGF-8 or partially purified latent platelet TGF-8. After a 12-h coincubation at 4 "C to allow any complex formation to occur, the pure active TGF-8 all migrates with the azM peak at 0.24 M NaCl (panel A ) . By contrast all of the partially purified latent platelet TGF-8 migrates as a distinctpeak centered around 0.34 M NaCl, with none being recoveredat the position of azM peak (panel B ) . This indicates that the platelet latent TGF-/3 cannot form a complex with serum azM just as cannot it with platelet azM. However, if the partially purified latent TGF-8 is activated by transient acidification prior to incubation with the azM, then 43% of the TGF-8activity is recovered in the azMpeak and 57% is recovered in the latent TGF-j3 peak (panel C). This suggests that it is only when the low molecular weight form of TGF-8 is released from the platelet latent complex by activation that itcan bind to aZM. The relative distribution of TGF-8 between the two peaks depends on the relative concentrations of azM and latent TGF-8 (data not shown). Thus, there is competition between the reformation of the latent complex and formation of a new complex betweenthe

A

B Ami-

Ad-TGFB

I

TGFP B S

-S +- + - + -+ -+ ++ + + + ++ ++ + ,

Ami-

TGFB PZ67-278 " 051

+

- + -

axm.

axm 1

2

3

4

5

1

2

3

4

5

FIG.9. Western blot analysis of latent TGF-@complexes in cellular-conditionedmedia. Concentrated conditioned media were prepared as described under "Experimental Procedures." A, all samples were cross-linked with 1 mM BS' prior to electrophoresis. Lanes had conditioned media from the following celltypes: human fibrosarcoma HT1080 ( l a n e I ) ; human rhabdomyosarcoma RD ( l a n e 2); rat epithelial line RCE ( l a n e 3); Fischer rat 3T3 fibroblasts transfected with the activated H-ras oncogene isolated from the human bladder carcinoma line E 3 ( l a n e 4). Lane 5 had platelet secretate for comparison. Blots were probed with anti-TGF-j3 with or without added TGFj3 as indicated, to demonstrate band specificity. B, conditioned medium from the human lung adenocarcinoma line A549was crosslinked where indicated, and blots were probed with anti-TGF-8 antiserum or anti-precursor peptide P267-279 antiserum.

activated TGF-8 and azM. (The azM is not saturated for TGF-8 in this experiment since in panel A, the same amount of aZM boundfive times as muchpurifiedTGF-8.) This suggests that acid activation of the latentcomplex is reversible under certain conditions.

The Latent Form of TGF-8 Secreted by Cultured Cells The Western blots in Fig. 9 show that all five of the cell types analyzed secrete a high molecular weight latent TGF81 complex similar to that secreted by platelets. For the human fibrosarcoma HT1080,the human rhabdomyosarcoma RD and the ratepithelial line RCE, the TGF-/3 latent complex appeared as a broad band of -215-245 kDa in cross-linked samples, whereasthe Ha-rastransfected Fischer rat 3T3 cells secreted a slightly lower molecular mass complex of-215 m a . This may reflectheterogeneity in glycosylation patterns. The HT1080-conditioned media had an additional TGF-8specificband at 98 kDa.Since the anti-TGF-8antiserum does not recognize TGF-82, these specificbands all represent TGF81 complexes. The same high molecular weight bands were recognized by the anti-precursor antibody P46-56 (data not ELUTION VOLUME Iml) FIG.8. Chromatographic analysis of complex formationbe- shown). Panel B shows that the latent complex in the conditween a*-macroglobulin and latent oractive platelet TGF-@. tioned medium of the human lung adenocarcinomaline A549 Partially purified platelet latent TGF-j3 complex was prepared by behaves identically to the platelet latent complex. Thus, the pooling fractions eluting between 0.33-0.44 M NaCl from Mono Q high molecular weightTGF-8 species is only detectable after column runs such as that in Fig. 7 and concentrating the pooled chemical cross-linking with BS', and the anti-precursor anmaterial by ultrafiltration using a YM-30 membrane. This partially tibody P267-279 detects the identical 220-235-kDa band in purified material contained nodetectable endogenous a2M. 430 pg of pure human serum anMwas mixed with either 180 ng of high pressure cross-linkedsamples and aslightly lower molecular massband liquid chromatography purified active human platelet TGF-j3 ( A ) ,or (-215 kDa) in untreated samples. The other bands in the with 26 ng of TGF-j3 equivalents of partially purified human platelet samples probed withP267-279 antibody are nonspecific (data latent TGF-j3 that was eitheruntreated ( B ) or was activated by not shown). transient acidification prior to addition to the anM (C).Samples were

IC

incubated overnight a t 4 "Cto allow complex formation to occur and were then analyzed by chromatography on aMono Q anion exchange column as described in the legend to Fig. 7. Samples A and B had added salt so that theionic strength was identical in all samples. The single arrow indicates that elution position of azM (0.24 M NaCI), and the double arrow indicates the elution position of the platelet latent TGF-j3 complex (0.34 M NaCI). The a2M peaks have been omitted from the figure for the sake of clarity, but in every case the a2M was detectable only in fractions 21 and 22.

DISCUSSION

We have shown that human platelets secrete TGF-8 in a biologically latent form. Thus, not only is TGF-8 stored latent (16), but it is delivered to the wound site in this form when platelets degranulate. The latentform comprisesactive TGF8, noncovalently associated with sequences correspondingto the remainder of the TGF-/3 precursor and a third unknown

Latent Transforming Growth Factor-P from Human Platelets component, giving a 220-235-kDa complex. The biochemical basis of the latency appears to be that the TGF-P receptorbinding site and other TGF-@ epitopes are masked, suggesting that active TGF-@may be surrounded by a protein “cage” in the latent form. Chaotropic agents and extremes of pH can activate the complex in vitro,presumably by disrupting its quaternary structure. While activation in vivomight occur in locally acidic microenvironments, recent work showing that proteases such as cathepsin D and plasmin can activate the complex in vitro (28) suggests the physiological activation mechanism could involve limited proteolysis. Although human platelets contain only a single form of TGF-b, TGF-Pl, a second closely related form, TGF-@2, hasbeen identified in porcine platelets, bovine bone, and conditioned medium from human glioblastoma cells and human prostatic adenocarcinoma cells (29-32). It will be important to establish whether TGF-@2is also latent and activated by similar mechanisms, or whether this might be a site of differential regulation of the two forms. Little is known about the biosynthesis of TGF-P, but our data indicate that, at least in platelets, minimal proteolytic processing of the precursor occurs beyond cleavage to release mature TGF-P, despite the presence of two potential tryptic sites inthe remainder of the precursor (15). The present work also shows that this portion of the precursor has complex N linked carbohydrate at at least two of the three potential glycosylation sites and that it may be covalently linked to a third protein via disulfide bonds prior to secretion. The identity of the third component in the complex is unknown, but it could play a role in the biosynthesis as well as conferring latency. Thus, by analogy with the EGF- and NGF-binding proteins (25, 261, this component might be a processing protease that remains stoichiometrically associated with the substrate after cleavage. However, this component does not appear to be immunologically related to the EGF-binding protein or the glandular kallikrein family, in agreement with recent suggestions that glandular kallikreins may not be the main physiological processors of bioactive growth factors (33). A new class of 70-kDa processing proteases, specific for dibasic residues has recently been described (34), and the third component might be a member of this family. Alternatively, the protein might be involved in directing the correct folding of the nascent TGF-@precursor, or in “molecular chaperoning” to prevent aggregation of the highly hydrophobic TGF-@ molecule (35). A role for the 70-kDa members of the heat shock family of proteins in this type of refolding process has been proposed (36). Obviously, further purification of the latent complex will benecessary for identification of the third component and analysis of any intrinsicenzymic activity. We have previously shown that thebiologically latent form of TGF-@found in serum is TGF-@bound to aZM (21) and thus is different from the latent form secreted by platelets. This presents a paradox, since the majority of the TGF-p found in serum presumably derives from platelet degranulation. In thepresent work we have shown that the latentform of TGF-@secreted by platelets will not bind to either serumor platelet-derived a2M. However, if the platelet latent TGF@ is firstactivated by transient acidification, the released active TGF-8 does bind to a Z M . Thus, the observation that serum TGF-/3 is recovered as an a2M complex could be rationalized if the latent form of TGF-/3 secreted by degranulating platelets is activated duringclot formation. The active TGF-P would then bind to serum a 2 M , which is present in stoichiometric excess. Since plasmin has been shown to activate latentTGF-@ in vitro (28), one of the many serine proteases involved in the clotting cascade might well be ca-

7653

pable of activating latent TGF-@. This possibility is currently under investigation. The latent form of TGF-@secreted by a variety of normal or transformed human and rodent cells in culture appears to be structurally similar, if not identical, to that secreted by platelets (see Fig. 9). We propose that the plateletlatent complex, and that secreted by cells in culture, are probably “delivery” complexes. These would extend the half-life of TGF-@in the extracellular milieu and ensurethat TGF-@ acts only on those target cells capable of activating the latent form, not indiscriminately on all surrounding cells. In contrast, the a2M/TGF-@ complex found in serum maybe a “clearance” complex, with azM scavenging any excess active TGF-P, thereby keeping TGF-@action locally confined to target tissues andpreventing systemic effects (21). Since the TGF-P receptor is essentially universally expressed (12), it is likely that any target restriction of TGF-@ action will be determined by the presence or absence of the activating mechanism for the latent form. Data from in vitro and in vivostudies using active TGF-P suggest that TGF-@ released by degranulating platelets a t wound sites might be involved in a paracrine fashion in monocyte and fibroblast recruitment, angiogenesis, and matrix deposition (13,37-39). However, this proposal should be re-evaluated in terms of whether the cell types involved are capable of responding to latent as opposed to active TGF-P, or whether there is some global activation of latent TGF-@throughout the wound site. Obviously, if the clotting process does indeed activate TGF-@ as we have suggested above, this would provide such a mechanism for extensive activation of TGF-@at the wound site. Similarly, to establish that TGF-P could be actingin an autocrine fashion on any cell type in vivo, it will be necessary to show not only that these cells secrete TGF-@ andrespond to exogenously added active TGF-P, but also that they can activate the latent form of TGF-@ thatthey secrete. We are currently purifying the latent form to use as a tool in elucidating the mechanism of this major control point in TGF-@ action. Acknowledgments-We thank Paul Isackson and Jack Pierce for reagents and expert advice relating to glandular kallikreins, Larry Mullen for invaluable assistance in the preparation of reagent antisera, and Anita Roberts for critical review of the manuscript. L. M. W. acknowledges the receipt of a NATO/SERC Overseas Postdoctoral Fellowship. REFERENCES 1. Sporn, M. B., Roberts, A. B., Wakefield, L. M. & de Crombrugghe, B. (1987) J . Cell. Bwl. 105,1039-1045 2. Roberts, A. B., Anzano, M. A., Lamb,L. C., Smith,J. M. & Sporn, M. B. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 53395343 3. Sporn, M. B., Roberts, A. B., Wakefield, L. M. & Assoian, R. K. (1986) Science 233, 532-534 4. Pircher, R., Lawrence, D. A. & Jullien, P. (1984) Cancer Res. 44, 5538-5543 5. Lawrence, D. A., Pircher, R., Kryceve-Martinerie, C. & Jullien, P. (1984) J. Cell. Physiol. 121, 184-188 6. Krychve-Martinerie,C., Lawrence, D. A., Crochet, J., Jullien, P. & Vigier, P.(1985) Znt. J. Cancer 35, 553-558 7. Lawrence, D. A., Pircher, R. & Jullien, P. (1985) Biochm. Biophys. Res. Commun. 133,1026-1034 8. Tucker, R. F., Shipley, G. D., Moses, H. L. & Holley, R. W. (1984) Science 226, 705-707 9. Shipley, G . D., Pittelkow, M. R., Wille Jr., J. J., Scott, R. E. & Moses, H. L. (1986) Cancer Res. 46, 2068-2071 10. Carr, B. I., Hayashi, I., Branum, E. L. & Moses, H. L. (1986) Cancer Res. 46,2330-2334 11. Sporn, M. B. & Roberts, A. B. (1985) Nature 313,747-751 12. Wakefield, L. M., Smith, D. M., Masui, T., Harris, C. C. & Sporn, M. B. (1987) J. Cell. Biol. 106,965-975

7654

Latent Transforming Growth Factor-0

13. 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, J. H. & Fauci, A. S. (1986) Proc. Natl. Acad. Sci. U. S. A . 83,4167-4171 14. Assoian, R.K., Komoriya, A., Meyers, C.A., Miller, D.M. & Sporn, M. B. (1983) J. Biol. Chem. 258,7155-7160 15. Derynck, R., Jarrett, J. A., Chen, E. Y., Eaton, D. H., Bell, J. R., Assoian, R. K., Roberts, A. B., Sporn, M. B. & Goeddel, D. V. (1985) Nature 3 1 6 , 701-705 16. Pircher, R., Jullien, P. & Lawrence, D. A. (1986) Biochem. Biophys. Res. Comrnun. 136,30-37 17. Masui, T., Wakefield, L.M., Lechner, J. F., LaVeck, M.A., Sporn, M.B. & Harris, C.C. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,2438-2442 18. Lundgren, S., Ronne, H., Rask, L. & Peterson, P. A. (1984) J. Biol. Chem. 259,7780-7784 19. Laemmli, U. K. (1970) Nature 277,680-685 20. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354 21. O’Connor-McCourt, M. D. & Wakefield, L. M. (1987) J. Biol. Chem. 262,14090-14099 22. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 23. Ganguly, P. (1969) Biochim. Biophys. Acta 1 8 8 , 78-88 24. Fanger, B. O., Wakefield, L. M. & Sporn, M. B. (1986) Biochernistu 25,3083-3091 25. Taylor, J. M., Cohen, S. & Mitchell, W. M. (1970) Proc. Natl. Acad. Sci. U. S. A. 6 7 , 164-171

from Human Platelets

26. Berger, E. A. & Shooter, E.M. (1977) Proc. Natl. Acad.Sci. U. S. A. 74, 3647-3651 27. Isackson, P. J., Silverman, R.E., Blaber, M., Server, A. C., Nichols, R. A., Shooter, E. M. & Bradshaw, R. A. (1987) Biochemistry 26,2082-2085 28. Keski-Oja, J., Lyons, R. M.& Moses, H. L. (1987) J.Cell Biochem. Suppl. 11A, 60 29. Cheifetz, S., Weatherbee, J. A., Tsang, M. L-S., Anderson, J. K., Mole, J. E., Lucas, R. & MassaguB, J. (1987) Cell 48,409-415 30. Seyedin, S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H. & Graycar, J. (1987) J. Biol. Chem. 262,1946-1949 31. Wrann, M., Bodmer, S., de Martin, R., Siepl, C., Hofer-Warbinek, R., Frei, K., Hofer, E. & Fontana, A. (1987) EMBO J. 6,16331636 32. Ikeda. T.. Lioubin. M. N. & Marauardt. H. (1987) Biochernistrv 26,’2406-2410 ’ 33. Isackson. P. J.. Dunbar. J. C. & Bradshaw. R. A. (1987) . . J. Cell. Biocheh. 33; 65-75 . 34. Parish, D. C., Tuteja, R., Altstein, M., Gainer, H. & Loh, Y. P. (1986) J. Biol. Chem. 2 6 1 , 14392-14397 35. Ellis, J. (1987) Nature 3 2 8 , 378-379 36. Pelham, H. R. B. (1986) Cell 46,959-961 37. Postlethwaite, A. E., Keski-Oja, J., Moses, H. L. & Kang, A. H. (1987) J. Exp. Med. 1 6 5 , 251-256 38. Wahl, S. M., Hunt, D. A., Wakefield, L. M., McCartney-Francis, N., Wahl, L. M., Roberts, A.B. & Sporn, M.B. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 5788-5792 39. Sporn, M. B., Roberts, A. B., Shull, J. H., Smith, J. M., Ward, J. M. & Sodek, J. (1983) Science 2 1 9 , 1329-1331 ,

.

I