Latent Forms of Transforming Growth Factor-/? (TGF/?) Derived from Bone Cultures: Identification of a Naturally Occurring 100-kDa Complex with Similarity to Recombinant Latent TGF/?

Lynda F. Bonewald, Lalage Wakefield, R. O. C. Oreffo, Alda Escobedo, Daniel R. Twardzik, and Gregory R. Mundy Department of Medicine Division of Endocrinology and Metabolism University of Texas Health Science Center San Antonio, Texas 78284-7877 National Cancer Institute National Institutes of Health (L.W.) Bethesda, Maryland 20892 Bristol-Meyers/Squibb (D.R.T.) Seattle, Washington 98121

Transforming growth factor-/? (TGF/?) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF/? homodimer from this latent complex is necessary for TGF/? to exert effects on target cells. Thus, the nature of the latent complex and the mechanisms responsible for TGF/} release are the key to understanding TGF/? actions. We have found that murine calvarial bone cultures secrete multiple latent forms of TGF0. Using analytical chromatography and Western blot analysis, we have compared bone latent TGF/? with the previously characterized latent complex present in platelets and with simian TGF/? precursor, which is stably expressed in a latent form by Chinese hamster ovarian (CHO) cells. A major component of the bone material appears to be a latent complex of 100 kDa, consisting of mature TGF/? (25-kDa homodimer) noncovalently associated with the remainder of the TGF/? precursor proregion (75-kDa homodimer). Like the recombinant TGF/? precursor, it elutes from a Mono-Q fast pressure liquid chromatography anion exchange column at 0.2 M NaCI and shows a very similar banding pattern on Western blots. Thus, this bone complex closely resembles recombinant TGF/? precursor expressed in a latent form by CHO cells and differs from the naturally occurring platelet complex, which has an additional 135-kDa binding protein that is bound through disulfide bonds to the precursor proregion. Western blot analysis also in-

dicates that, like CHO cells, which express recombinant TGF/? precursor, but unlike other cell types, the bone cultures secrete detectable amounts of uncleaved TGF/? precursor. The bone calvarial culture is the first example of a naturally occurring system that expresses the 100-kDa latent TGF/? complex. (Molecular Endocrinology 5: 741-751, 1991)

INTRODUCTION

Biologically active TGF/?-1 is a homodimer with a mol wt of 25 kDa. This molecule has dramatic effects in a number of cell systems in vitro (for review, see Ref. 1) and in vivo (2, 3). TGF/? may function by modulating or enhancing the effects of other growth factors. TGF/? is produced by cells in a biologically latent form, which can be activated by changes in pH (4, 5, 6), by addition of dissociating agents, by incubation with enzymes such as plasmin (7, 8), and by stimulated cells, such as osteoclasts, which have been pretreated with retinol or bone particles (9) and interferon-treated macrophages (10). Several different latent forms of TGF/? have been identified. The latent form of TGF/? found in serum is an a2-macroglobulin-TGF/? complex (11) and may represent a clearance form. Platelets contain a 235-kDa latent complex composed of mature TGF/31 (25-kDa dimer) noncovalently associated with the remainder of the TGF/?1 precursor proregion (75-kDa dimer), which,

0888-8809/91 /0741 -0751 $03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society

741

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MOL ENDO-1991 742

in turn, is bound through disulfide bonds to a binding protein of 135 kDa (12,13). Recombinant human TGF0 expressed by Chinese hamster ovarian (CHO) cells is secreted by these cells in a latent form of 100 kDa, which lacks the 135-kDa binding protein of the platelet complex (14). This suggests that the 135-kDa binding protein is apparently not required to render TGF/3 latent. TGF/3 appears likely to be a key factor in normal bone cell function. The major source of stored TGF/3-1 in the body is the bone matrix (15, 16). Preliminary studies have suggested that forms of latent TGF/3 in this site are likely to be heterogeneous (17). TGF/3 has marked effects on both osteoblasts and osteoclasts (for review, see Ref. 18). We have previously shown that TGF/3 activity in bone cultures can be increased by boneresorbing hormones (19), suggesting that TGF/3 may serve as a coupling factor, i.e. an osteoblast stimulator released during the resorption process. Since bone is the major source of TGF/3 in the body, and TGF/3 is likely to be involved in the bone-remodeling process, we sought to determine the nature of the latent TGF/3 complex produced by bone cultures. We found that TGF/3 is produced by bone cultures as a complex mix of latent forms. One form of latent TGF/3 is a 100-kDa complex that is different from the latent TGF/3 present in platelets, but more closely resembles the latent TGF/3 produced by CHO cells transfected with simian TGF/31. Bone is the only tissue so far described that contains this type of latent complex. This suggests that the 100-kDa complex may have properties uniquely suited to its role in bone physiology.

RESULTS Comparison of Bone, Platelet, and Recombinant Latent TGF/3 Complexes on Analytical Ion Exchange Chromatography Previous studies had shown that bone organ cultures release TGF/3 in a latent state (20). To characterize this form of latent TGF/3, we compared the latent TGF/3 complex present in bone culture medium with the latent TGF/3 complexes present in platelet secretate and in conditioned medium harvested from CHO cells transfected with simian TGF01. Bone and platelet latent complexes were enriched by fractionation on Sephadex G-200. Protein fractions greater than 70 kDa were pooled and used as starting material for subsequent experiments. There was no evidence of any lower mol wt forms of TGF/3 (data not shown). We compared the properties of recombinant latent TGF/3 and the G-200enriched bone and platelet fractions by chromatography on Mono-Q ion exchange columns using fast pressure liquid chromatography (FPLC). The platelet latent TGF/3 complex eluted as a major peak at 0.3 M NaCI (Fig. 1 A). The bone-derived latent TGF/3 complex eluted as two distinct peaks at 0.2 M NaCI (peak I) and 0.3 M NaCI (peak 2). TGF/3 activity was approximately equally distributed between these two peaks (Fig. 1B). The recom-

Vol 5 No. 6

binant TGF0 precursor obtained from CHO cell culture supernatants was also applied to this ion exchange column. It eluted in a peak at 0.2 M NaCI (Fig. 1C). Considerably more TGF/3 biological activity was detectable in the recombinant TGF/3 material from CHO-conditioned medium than from the platelet secretate or bone organ-conditioned medium. CHO-conditioned medium contained up to 0.5-1 Mg/ml bioassayable TGF/3, whereas platelet secretate contained 0.1-0.3 Mg/ml, and bone-conditioned medium contained 5-8 ng/ml. Gel Filtration Sizing of Bone-Derived Latent TGF/3 Complexes Each of these peaks of activity from the ion exchange columns was then chromatographed separately on a Superose 12 gel filtration column equilibrated in PBS to get an estimate of the complex molecular mass. The platelet latent complex which eluted as a single peak at 0.3 M NaCI, has a molecular mass of approximately 600 kDa (Fig. 2A). Essentially all bioassayable material in this peak was blocked by antibodies specific for TGF/31 (data not shown). The bone-derived latent complex that eluted from the ion exchange column in a similar manner as the platelet secretate at 0.3 M NaCI (peak 2; Fig. 2C) possessed a mass similar to the platelet material (600 kDa), with a minor peak of activity at 100 kDa. The bone-derived latent complex that eluted from the ion exchange column at 0.2 M NaCI (peak 1) was also chromatographed on the Superose 12 column. Biological activity was eluted in two peaks, one at 400-600 kDa and one at -100 kDa (Fig. 2D). When recombinant latent TGF/3 was chromatographed on this column, the major peak of activity eluted at 100 kDa (Fig. 2B). These results suggest that bone cultures secrete multiple latent forms of TGF/3, including one that is similar to the platelet complex and one that is more like the recombinant latent complex. Subtype Analysis of Bone-Derived Latent TGF/3 Three distinct subtypes of TGF/3 have been described in mammalian systems, two of which have been described in bone (TGF/31 and TGF/32). To determine the subtype of TGF/3 present in the latent complexes, we used an antibody specific for TGF/31 and a second antibody, which neutralizes TGF/31, TGF/32, and TGF/33, and examined the capacity of these antibodies to neutralize acid-activated TGF/3 in the mink lung assay. We tested the 100-kDa peak from the Superose 12 column of the 0.2 M NaCI bone-derived complex (peak 1) using these antibodies. We found that the antibody specific for TGF/31 completely inhibited this biological activity (Fig. 3). These results suggest that the 0.2-M NaCI peak (1) from the ion exchange column comprises TGF/31. However, peak 2 (0.3 M NaCI) from the Mono-Q ion exchange column of the bone-derived material could not be completely inhibited by antibodies for TGF/31, although it was inhibited by the antibody that blocked TGF/31, TGF/32, and TGF/33. This suggests

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743

Latent Forms of TGF/3 from Bone Cultures

Non-Reduced Conditions

Reduced Conditions

A. Uncleaved Precursor CHO

CHO

CHO

CHO

CHO

CHO

54kD (from A)

100kD

42kD I CHO

CHO

(from B)

CHO

CHO

~ CHO CHO

12.5kD B. Recombinant Latent CHO

75 + 25kD

1 ,s 1

s

I1.,

CHO

CHO

CHO

, 1

s s

s s CHO

1

CHO

i

Cleavage site Fig. 1. Both A and B Forms Are Produced by Transfected CHO Cells * , Cleavage site; 42 kDa, precursor proregion monomer; 54 kDa, noncleaved precursor monomer (contains TGF/3 monomer); 12.5 kDa, TGFj3 monomer; 100 kDa, precursor homodimer plus TGF/J homodimer; 75 kDa, latency-associated peptide or the dimer form of the TGF/31 precursor proregion; 25 kDa, mature TGF/3 homodimer.

that this particular fraction may contain TGF/31 and either or both TGF/32 and TGF03. Western Blot Analysis of Bone Latent TGF/J Complexes To characterize the bone latent complexes further, we compared them first to the platelet complex and then to the recombinant complexes by Western blot analysis, using antibodies specific for either the TGF/31 precursor proregion or mature TGF/3. Using a 3-12% gradient gel under nonreducing conditions, the platelet TGF/3 complex and the bone-derived latent complex derived from gel filtration chromatography were compared and showed a 235-kDa band for the platelet-derived material and a 100-kDa band for the bone material (Fig. 4). In the bone-derived latent complex, two nonspecific bands were seen in the lanes treated either with or without the antibodies. We also examined the bone-derived material eluting at 0.2 M NaCI (peak 1) from the Mono-Q ion exchange column using anti-TGF/3-1 antisera (Fig. 5, left panels). Again, a 100-kDa band was seen, whereas no clear band was seen when the 0.3-M NaCI fraction (peak II) was treated in the same manner. With the same sample, but using an antibody to the precursor portion of mo-

lecular TGF/31, the platelet material showed a band at 235 kDa, and a 100-kDa band was found in the material eluting at 0.2 M NaCI (peak I; Fig. 5, right panels). Again, no clear identifiable band could be seen in the bone material eluting at 0.3 M NaCI (peak II) from the ion exchange columns. When peaks I and II were compared using either antiTGF/3 or antiprecursor antibody, the 100-kD band was always stronger in peak I. Peak II may have been partially contaminated with peak I, because by mass determination studies, a small amount of the 100-kD material is present (Fig. 2C). According to blocking antibody experiments (Fig. 3), peak II contained both TGF/31 and TGF02 and/or TGF/33 latent complexes. This suggests that peak II contains two or more forms of latent TGF/3. This second peak has proved difficult to characterize, while peak I was more straightforward. This is probably due to the homogeneity of peak I and the heterogeneity of peak II. As the bone material appeared to be the same mol wt as recombinant TGF/3 precursor, a direct comparison was performed. Human (21), simian (22), and murine (23) TGF/31 are synthesized as a 390-amino acid precursor polypeptide. This precursor is proteolytically cleaved to yield the mature 112-amino acid monomer. Gentry et al. (14) have expressed high levels of recom-

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Vol 5 No. 6

MOL ENDO-1991 744

2.5

A. Platelet Latent Complex

2.0

1.5

0.50.0 . „,. '•"T

10

15 fraction # B. Bone Latent Complexes

20

25

30

1.00-

however, cross-linking the bone or recombinant material resulted in lighter bands, probably due to the loss of immunoreactive epitopes. In fact, cross-linking the recombinant material gave dimers, trimers, and tetramers. Therefore, cross-linking was not performed using the recombinant material (see Figs. 7 and 8). Using a 9% gel under nonreducing conditions, the recombinant TGF/3 precursor and bone-derived material that had previously been applied to Sephadex G-200 gel filtration were compared (Fig. 7). A 100-kDa band with a very similar migration pattern was seen in both the bonederived material and the recombinant material (see Fig. 1). Using a-TGF/3 antibody and reducing conditions, the bone material (peak I from ion exchange) produced a band very similar in migration to that seen with recombinant TGF/3 precursor (Fig. 8, left panels). A 54-kDa band was noted as well as a faint 12-kDa band correlating to the noncleaved precursor monomer and the TGF/3 monomer (see Fig. 1). This same bone material applied under the same conditions except using antiprecursor antibody as primary antibody resulted in two bands, 54 and 42 kDa, which correlates to the noncleaved precursor monomer and the precursor proregion monomer (see Fig. 1). Very similar bands were seen with the recombinant TGF/3 material. A comparison of the bone-derived latent complexes of TGF/3 with platelet, recombinant, and a2-macroglobulin latent forms has been compiled in Table 1.

•0.1

DISCUSSION 25

+-•-0.0 30

Fig. 2. Gradient Profile and Activity Profile of Platelet Latent TGF/3 G-200 Fraction (A), Bone Latent TGF/3 G-200 Fraction (B), and Recombinant TGF/3 Precursor (C) Applied to a Pharmacia Mono-Q Ion Exchange Column Using FPLC The platelet latent material elutes in a major peak at 0.3 M NaCI, whereas the bone material has two peaks of activity at 0.2 and 0.3 M NaCI. The recombinant precursor elutes at 0.2 M NaCI. No activity was eluted after 0.5 M NaCI. Buffer A, 20 miui Tris (pH 7.2); buffer B, 1.0 M NaCI in buffer A. 1 , TGF/3 activity.

binant simian TGF/3-1 in CHO cells. We used this protein in our studies. Supernatants from CHO cells contain a 90- to 110-kDa form of latent complex as well as the mature 24-kDa homodimer detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under nonreducing conditions (see Fig. 1). Under reducing conditions on SDS-PAGE, a 44- to 55-kDa band correlating with the noncleaved precursor, a 30to 42-kDa band correlating with the cleaved precursor, and a 12-kDa band which is the mature TGF/31 monomer were observed. We found cross-linking unnecessary for the bone and recombinant material, as neither dissociated under electrophoresis. Cross-linking the platelet material gives a sharper band, compared to noncross-linked material;

Our study shows that bone organ cultures release TGF/3 as several latent high mol wt complexes, and we have characterized one major form which is distinct from the platelet-derived latent complex. This bonederived complex behaves similarly to recombinant latent TGF/3 precursor on size separation and ion exchange columns, and on Western blotting using antibodies to both the TGF/3 precursor proregion and active TGF/3. The data suggest that, like the recombinant TGFjS precursor, the bone-derived complex consists of mature TGF/31 (25-kDa dimer) noncovalently associated with the remainder of the precursor proregion (75 kDa). Both recombinant and bone material differ from the latent complex secreted by platelets and other cells in culture, in that they lack the 135-kDa binding protein. Other cell types that have been analyzed to date secrete latent TGF/3 in a form similar to the platelet complex (12). Bone is the only tissue described so far in which the latent form consists of a 100-kDa complex. This may indicate a unique role for this type of latent TGF/3 complex in bone. Proteolytic processing of the 390-amino acid TGF/3 precursor to yield mature TGF/3 occurs at the dibasic cleavage site preceding Ala-279 at a dibasic Arg-Arg sequence. Little is known of the enzyme responsible for cleavage of the precursor (14), and cleavage does not lead to activation. We have denoted the reduced

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Latent Forms of TGF/3 from Bone Cultures

60

745

60 j

T

50--

50-

40--

40--

30-

30--

20--

20-•

10--

10--

18

12 14 Fraction Number

8

B. rTGF/? Precursor

10

12 14 Fraction Number T

60-r

60 j

C. Bone Peak II (0.3 M NaCl)

50-

"? 50--

40--

.2 40 +

i

D. Bone Peak I (0.2 M NaCl)

o u

o>

30>

20--

20-

10--

10--

08

gg gg qg

0 10

12 14 Fraction Number

16

18

8

10

12 14 Fraction Number

16

18

Fig. 3. A Superose 12 Gel Filtration Activity Profile of Platelet Material (A), Recombinant TGF/3 Precursor (B), Bone Peak II (0.3 wi NaCl; C), and Bone Peak I (0.2 M NaCl; D) Each fraction number equals retention volume in milliliters. Half-milliliter fractions were collected. The buffer was PBS.

form of noncleaved precursor the 54-kDa band, whereas the 42-kDa band is the reduced form of cleaved precursor or the proregion. As the same forms are observed in the bone material, the bone material is also probably secreted in cleaved and noncleaved forms. This is the first example of a natural cell type secreting unprocessed precursor. The unprocessed precursor does not have TGF/3 activity and cannot be acid activated (24). It is not known whether the uncleaved precursor can be processed extracellularly or whether this uncleaved precursor has some unique, as yet undetermined, biological activity. The cDNA sequence of TGF/3 suggests that there are three potential sites for N-linked glycosylation (25) in the precursor proregion, and the precursor has been found to contain carbohydrate sialic acid. Mature TGF/3 is not glycosylated. As both the reduced and nonreduced bands in the bone-derived latent complex migrated on SDS-PAGE in the same manner as the recombinant material, this suggests that the bone material is similarly glycosylated. Two of the three asparagine-linked carbohydrate chains in recombinant TGF/3 precursor contain mannose-6-phosphate residues (26), and the precursor has been shown to bind to the mannose-6-phosphate receptor (27). This may

also be true for bone-derived latent TGF/3. We have shown previously that isolated avian osteoclasts activate bone latent TGF/3, and since osteoclasts contain mannose-6-phosphate receptors (28), these cells may activate latent TGF/3 through binding to these receptors, followed by intemalization of the ligand-receptor complex. In addition to the 100-kDa complex, we have shown that other latent complexes are secreted by bone cultures. We have been unable to characterize definitively the fraction of bone-derived latent TGF/3 that elutes at 0.3 M NaCl from Mono-Q ion exchange and has a mass of 600 kDa, but its chromatographic properties are similar to those of the platelet latent complex. Antibody neutralization data suggest that other subtypes of TGF/3 in addition to TGF/31 may be present in this peak. Since the Western blot reagents are specific for TGF(81, this may explain why no bands corresponding to this material were visible in Fig. 5. The binding protein in the latent TGF/3 complex in other tissues has been shown to exist in two forms; a form has been described in fibroblasts of 170-190 kDa, and a separate form in platelets of 125-160 kDa. These differences in size may be due to alternative splicing or cell-specific proteolysis (29). The binding protein contains 16 epidermal growth factor-like repeats and hy-

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Vol 5 No. 6

MOL ENDO-1991 746

• I No addition CD Anti-TGF/? I ZZZ Anti-TGFjS I + TGF0 II

100-

?

80 + 60--

o

40 +

CM

20--

I PBS/BSA

I

400-600 kD 100 kD peak peak

TGF/S I 10 pM

MonoQ peak I

600 kD peak MonoQ peak II

Fig. 4. Reversibility by Anti-TGF^ Antibodies of CCL64 Growth Inhibitory Activity in Peak Fraction from Superose 12 Fractionation of Partially Purified Bone Latent Material from Mono-Q Peak I (0.2 M NaCI) and Peak II (0.3 M NaCI) • , No antibody; D, addition of TGF/M-specific neutralizing antibody; H, addition of antibody that neutralizes TGF/J1, TGF/?2, and TGF/33. Samples were tested in duplicate with less than 5% deviation.

•anti-TGF/3Blocking i Peptide

Bone

Platelet I _

235kd> -200

-97

IOOkd>

-66 -43 25kd>

25kd>

Fig. 5. Western Blot Using Affinity-Purified Antibody to TGF01 under Nonreducing Conditions Samples tested are cross-linked bone latent TGF/3 from Sephadex G-200 gel filtration column and cross-linked partially purified platelet latent TGF/3 (3-10% SDS-PAGE). The mol wt of standards are 200, 97, 66, and 43 kDa. - , Absence of TGF|81; +, presence of TGF01.

droxylated asparagine residues (29, 30). In addition, it contains an RGD sequence and a sequence of eight amino acids identical to a sequence in the proposed cell-binding domain of the laminin B2 chain, which may be responsible for interactions with integrins and other membrane molecules. As the latent complex in boneconditioned medium lacked this binding protein, it may be absent in secreted forms of latent complex, but may be present in matrix or storage forms of latent TGF/3. This is presently under investigation. Multiple latent forms of TGF/3 have also been identified in bone matrix by others. Jennings and Mohan (17) have extracted bovine bone matrix with EDTA and identified several latent forms of TGF/3. Four discrete peaks at 0.22, 0.25, 0.35, and 0.42 M NaCI on MonoQ anion exchange chromatography were detected. Plasmin treatment only activated the 0.35- and 0.42-M fractions. The 0.35-M fraction upon reduction and reverse phase HPLC yielded TGFj81, TGF/32, and an unknown TGF/3. Their data suggest multiple latent forms of TGFjS in bone matrix. It is possible that some of these latent forms contain the binding protein. In the present study we have identified a latent form of boneconditioned medium that possessed characteristics similar to those of the platelet complex. These characteristics are elution from ion exchange with 0.3 M NaCI, a mass of 600 kDa, and activity of the TGF/31 isotype. However, we were not able to confirm identity to the platelet complex by Western blotting. Therefore, this complex could contain the binding protein and may represent a bone matrix form of TGF/3. The possibility

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Latent Forms of TGF/3 from Bone Cultures

747

r— anti-TGF/3—| Blocking Peptide

Peak 1

anti-precursor

PeakD

Peak I

Platelet

PeakD

r_

235kd> -97 100 kd>

100 kd>

-66 -43 -31

-97 -66 -43 -31 -21.5 - 14.5

-21.5 -14.5

Fig. 6. Left Panel, Western Blot Using Affinity-Purified Anti-TGF/31 under Nonreducing Conditions Samples tested are cross-linked bone peak I, which elutes from ion exchange at 0.2 M NaCI, and cross-linked bone peak II, which elutes at 0.3 M NaCI on ion exchange (4-12% SDS-PAGE). - , Absence of TGF/31; +, presence of TGF01. Right panels, Western blot using antibody that recognizes the precursor region of TGF/31 under nonreducing conditions. Samples tested are cross-linked platelets, cross-linked bone peak I (0.2 M NaCI), and cross-linked bone peak II (0.3 M NaCI; 4-12% SDS-PAGE). The mol wt of standards are 97, 66, 43, 31, 21.5, and 14.4 kDa, prestained and reduced. - , Absence of precursor peptide 244-267; +, presence of precursor peptide 244-267.

•anti-TGF£Blocking Peptide

Bone

Recombinant

HI

I"

'm 100 kd>

m

IOOkd>|

-no -84

-47

-33 -24

Fig. 7. Western Blot Using Affinity-Purified Anti-TGF/31 on a 9% SDS-PAGE under Nonreducing Conditions Samples tested are bone material from G-200 column and recombinant TGF/3 precursor from CHO-conditioned medium. Neither the bone nor the recombinant material was crosslinked. The mol wt standards are 100, 84, 47, 33, and 24 kDa, prestained and reduced. - , Absence of TGF/31; +, presence of TGF/31.

exists that the 100-kDa bone-derived latent TGF/3 in organ culture medium is a secreted form and different from the latent TGF0 complex present in bone matrix. The 100-kDa bone-derived material we studied obviously lacks the platelet-binding protein. This binding

protein is not necessary for latency, but may have other functions. The binding protein possesses several calcium-binding sites (29). Bone matrix TGF/3 may contain this calcium-binding protein, which could function in attachment and anchorage in bone. Investigation of this possibility is currently underway in our laboratory. In a previous study we have shown that there is an increase in active TGF/3 in conditioned medium harvested from bone cultures during bone resorption (19) and that retinol-treated avian osteoclasts activate bonederived latent TGF/3 (9). Ideal conditions for TGF/3 activation in bone occur in the acidic microenvironment under the ruffled border of the osteoclast during bone resorption (31-33). It is, therefore, possible that latent TGF/3 stored in bone matrix or released by bone cells becomes activated at this site during the bone resorption process. Since TGF/3 has been shown to have stimulatory effects on osteoblastic cells, such as increased production of matrix-associated proteins (19, 34), and inhibitory effects on osteoclasts in a variety of in vitro systems (35-37), activation of TGF0 during bone resorption may, therefore, present one of the mechanisms that link bone resorption to new bone formation. The regulation of the release of active TGF/3 from its various latent forms may be vital for understanding the local control of TGF/3 activity. Identification of this 100-kDa naturally occurring latent form of TGF/3 with characteristics of the TGF,8 precursor in bone validates further examination of recombinant TGF/3 precursor for biological effects both in vitro and in vivo. Use of recombinant TGF/3 may have potential therapeutic application in relation to bone remodeling and formation. MATERIALS AND METHODS Acid Activation of Latent TGF/3 in Samples In all experiments latent TGF/3 fractions from gel filtration and ion exchange columns (see below) of bone-conditioned me-

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Vol 5 No. 6

MOL ENDO-1991 748

-anti-TGF/3Blocking Peptide

Bone

ant i-precursor Bone

Recombinont ~

-100

-•*

-84 54kd>

1

Recombinant

54kd>

-47 -33

-100 -84

54kd>.—•

54kd>

42kd>

42kd>

-47

-33

-24

-24

Fig. 8. Left Panels, Western Blot Using Affinity-Purified Anti-TGF,81 (9% SDS-PAGE) under Reducing Conditions Samples tested are bone material peak I, which elutes from ion exchange at 0.2 M NaCI, and recombinant TGF/3 precursor from CHO-conditioned medium. - , Absence of TGF/31; +, presence of TGF/31. Right panels, Western blot using antiprecursor antibody under reducing conditions. Samples tested are bone material peak I (0.2 M NaCI) and recombinant TGF/3 precursor (9% SDSPAGE). The mol wt of standards are 100, 84, 47, 33, 24, and 16 kDa, prestained and reduced. - , Absence of precursor peptide 244-267; +, presence of precursor peptide 244-267.

Table 1. Comparison of Bone Material with Other Latent Forms of TGF/3 Sample

Platelet Recombinant Calvarial Bone Peak 1 (0.2 M Nad) Calvarial Bone Peak 2 (0.3 M NaCI) «2-Macroglobulina

NaCI (Elution Molarity from Ion Exchange)

Western Blot (kDa)

600 -100 450-600 and -100 600

0.3 0.2 0.2

235 100 100

0.3

?

400 or >600

0.2

185

Mass (Superose 12; kDa)

TGFjS Subtype

1 1 1 1,2,3 ND

?, Could not be determined. ND, Not done. " As reported by O'Connor-McCourt and Wakefield.

dium, platelet secretate, and CHO cell-conditioned medium were activated by adding 1 N HCI to the sample to a final pH of 2, followed by return to pH 7.4 by the addition of 1 N NaOH. In experiments in which acid-activated and nonactivated samples were assayed, equal amounts of 1 N NaCI were added to the nonactivated samples and to all TGF/3 standard dilutions to avoid differences in volume and osmolarity. CCL64 Assays The mink lung assay was performed as described by Danielpour et al. (38). Biological activity was assessed by growth inhibition of the mink lung epithelial line CCL64 obtained from American Type Culture Collection (Rockville, MD). Growth inhibition was measured as the decrease in incorporation of the thymidine analog 5-[125l]iodo-2-deoxyuridine. Specificity for TGF/31 was tested using a TGF/31 affinity-purified antiturkey antibody (39) and an antibody (R & D Systems, Minneapolis, MN) that neutralizes TGF/31, TGF02, and TGF/33. Alkaline Phosphatase Microassay This assay was performed essentially as described previously (40,41). ROS 17/2.8 cells were plated at 104 cells/well in 10% Dulbecco's Modified Essential Medium (Flow Laboratories,

MacLean, VA) for 24 h in 5% CO2 at 37 C, after which time medium was removed, and the factors and samples were applied. Fractions were tested in duplicate at various dilutions, as indicated in the text. The cells were incubated for an additional 48 h, after which time the medium was aspirated, the cells were washed twice with PBS, and 100 Ml/well 0.05% Triton were added. The microtiter plate was freeze-thawed twice, each well was mixed with a Titre-Tek pipettor (Flow Laboratories), and an additional 100 ^l 0.05% Triton were added per well. Approximately 10-20 /xl of the total cell lysate were used for the protein determination, and 5-10 ^l were used for detection of alkaline phosphatase activity. Specific alkaline phosphatase activity was determined as follows. The protein standard, consisting of 1-4 ^g human immunoglobulin G (Bio-Rad, Richmond, CA)/160 ^l-well, was assayed in triplicate in a 96-well plate, with percent Triton X100 taken into account. Bio-Rad Protein Reagent (40 Ml/well) was then added and immediately mixed. The plate was read at 600 nm on a M. A. Bioproducts EIA microtiter plate reader (Walkersville, MD) within 5-30 min after mixing. Alkaline phosphatase substrate and standard were made according to the protocol of Majeska et al. (42). The standard, p-nitrophenol phosphate (Sigma, St. Louis, MO), consisted of 2-14 nmol/100 jitl-well AMP buffer in triplicate in a 96-well microtiter plate. The aminomethylpropanol (AMP) buffer is

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749

Latent Forms of TGF/3 from Bone Cultures

composed 0.5 M 2-amino-2-methylpropanol, pH 10, and 8 mM p-nitrophenol phosphate-2 mM MgCI. Stop solution (0.5 N NaOH; 100 //I/well) was added, and the plate was read at 410 nm. From linear regression analysis of the protein and p-nitrophenol standards, the mean specific activity of each fraction sample was calculated. The alkaline phosphatase activity is expressed as specific activity (nanomoles of alkaline phosphate per ng protein/min) and was converted to equivalents in TGF/3 activity. A TGF/3 standard curve was performed in each assay using 10 ng/ml purified TGF/3 (Collagen Corp., Palo Alto, CA) and diluted 1:2 in duplicate. All calculations were processed using a program written specifically for this assay on an IBM PC. Blocking antibody specific for TGF/31 (Collagen Corp.) was used at a 1:1000 dilution to verify that all activity detected was due to TGF/31. Bone Organ Cultures and Collection of Bone Conditioned Medium Calvarial bone-conditioned medium was collected as described previously (20). Briefly, frontal and parietal calvaria from 4-dayold mice (ICR Swiss) were cultured in serum-free BGJb medium (Gibco, Grand Island, NY)-0.1% BSA on steel grids at the interface between medium and air. The calvaria were preincubated for 24 h; the medium was then changed, and conditioned medium was collected after an additional 48 h. Approximately 500 ml conditioned medium were collected for each isolation. Collection of Platelet Secretate Platelet degranulation was performed essentially as described by Wakefield et al. (12), with the following modifications. Outdated human platelets (South Texas Regional Blood Bank, San Antonio, TX) were centrifuged, washed twice in Dulbecco's PBS, resuspended at 3-4 x 109 platelets/ml, and induced to degranulate by the addition of 1 U/ml human thrombin (Sigma) for 10 min at 37 C, then the reaction was stopped on ice, at which time BSA (Sigma) and protease inhibitors phenylmethylsulfonylfluoride (0.005%), aprotinin (0.001%), leupeptin (0.002%), and pepstatin (0.0005%; Sigma) were added. The material was centrifuged at 2500 x g for 1 h at 4 C, and the supernatant was used for further experiments. Latent Recombinant TGF01 Recombinant latent TGF/3 was obtained from CHO cells transfected with simian precursor TGF/31 (14). Conditioned medium was used for Western blotting, ion exchange, and gel filtration studies. Conditioned medium from the CHO expression system contains the 100-kDa unprocessed TGF/3 precursor and a complex comprising mature TGF/3 (25-kDa dimer) noncovalently associated with the remainder of the precursor proregion (75-kDa dimer; see Fig. 1). Only the noncovalent cleaved 100kDa complex is a true latent complex that can be acid activated to release active TGF/3. Seven to 20% of the recombinant precursor can be activated by acidification (14, 24). Analytical FPLC Gel Filtration for Mass Determination Gel filtration was performed using a Superose 12, 10 x 300mm column (Pharmacia, Piscataway, NJ) equilibrated with PBS. Each fraction (1.0 ml) was acid activated and tested for TGF/3 activity using the CCL64 assay with and without blocking antibody specific for TGF/31 and an antibody that recognizes TGF/91, TGF/32, and TGF/33 (R & D Systems).

13,500 mol wt cut-off membrane (Spectropor; Scientific Products, McGaw Park, IL), lyophilized, and reconstituted in the same buffer. The neutral reconstituted conditioned medium was applied to a Sephadex G-200 column (2.5 x 75 cm; Pharmacia), which had been equilibrated with 50 mM NH4HCO3, pH 7.2, and fractions were eluted at a flow rate of 5 ml/h. Fractions of 9.1 ml were collected, lyophilized, reconstituted to 0.5 ml in PBS, and sterilized by filtration through a 0.22-Mm filter (Spin-X, Costar, Cambridge, MA), then aliquots of each fraction were tested in the alkaline phosphatase microassay with prior acid activation. The column was calibrated using BSA (68 kDa), ovalbumin (43 kDa), chymotrypsinogen (23 kDa), and ribonuclease (14 kDa; Bio-Rad). Ion Exchange Chromatography The active fractions from gel filtration were applied to a MonoQ anion exchange column (Pharmacia). Both an analytical (5 x 50 mm) and a preparative (10 x 100 mm) column were used. The sample was applied in 20 mM Tris buffer, pH 7.2, maintained for 5 min before elution at 1 ml/min over 60 min with a linear gradient of 0-0.6 M NaCI 20 mM Tris buffer in 1ml fractions for the analytical column. The gradient was extended to 3 h for the preparative column, and 7-ml fractions were collected. All fractions were dialyzed against 50 mM NH4HCO3 (pH 7.2), acid activated, and tested at three or four dilutions for TGF/3 activity in the alkaline phosphatase microassay. Cross-Linking Protocol Ten microliters of disuccinimidyl suberate (50 mM; Pierce, Rockford, IL) in dimethylsulfoxide (Sigma) were added to each sample (200 /*!) for 1 h at 4 C, after which time the samples were subjected to SDS-PAGE. Electrophoresis and Western Blotting SDS-PAGE was performed according to the method of Laemmli (43), using a Mini-Protean electrophoresis apparatus (BioRad). Material was applied to either a linear or a gradient SDSpolyacrylamide gel, as noted in the text, which were then transblotted onto nitrocellulose membrane (Bio-Rad) using Tris glycine buffer (25 mM Tris base and 0.2 M glycine; Sigma) and 20% methanol (Fischer, Pittsburgh, PA) at 20 mamp overnight. The current was increased to 100 mamp for 1 h before removing the membrane. The nitrocellulose was blocked with 5% BSA (0.1% Tween) in PBS for 1 h, then blotted with affinity-purified antibody specific (1:1000) for TGF/31 (12) or with antibody that recognizes the TGF/31 recombinant precursor region 244-267 (12) (1:50), and an identical membrane was blotted against TGF/3 antibody plus 1 ^g/ml TGF/3 or precursor antibody plus 10 ng/m\ peptide 244-267 to rule out nonspecific binding of primary antibody. Secondary antibody was alkaline phosphatase conjugated goat antirabbit (Zymed, San Francisco, CA) at a dilution of 1:2000. The blots were developed using a kit that contains 5-bromo-chloro-3-indolyl phosphate (0.016%) and nitroblue tetrazolium (0.03%) in Trisbuffered saline (Bio-Rad). The mol wt markers used were prestained and reduced with mol wt of 97, 66, 43, 31, 21.5, and 14.4 kDa (Diversified Biotech, Newton Centre, MA) for the platelet-containing Western blots. Aprotinin (200 kDa; Sigma) was also used as a mol wt marker. The mol wt markers used for Western blots containing the recombinant material were also prestained and reduced, but with mol wt of 110, 84, 47, 33,24, and 16 kDa (Bio-Rad).

Preparative Gel Filtration

Acknowledgments

Conditioned medium and platelet secretate were dialyzed against 0.05 M ammonium bicarbonate buffer, pH 7.2, using a

We are grateful to Gloria Peche for her secretarial expertise, and to Mary Beth Kester for expert technical assistance.

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Vol 5 No. 6

MOL ENDO-1991 750

Received December 17, 1990. Revision received February 14,1991. Accepted March 12, 1991. Address requests for reprints to: Dr. Lynda F. Bonewald, Department of Medicine, Division of Endocrinology and Metabolism, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7877. This work was supported by Grants AR-39357, DE-08569, AR-39529, and AR-07464 from the NIH.

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Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta.

Transforming growth factor-beta (TGF beta) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF beta h...
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