The EMBO Journal vol.10 no.5 pp.1091 - 1101, 1991

A role of the latent TGF-3 1-binding protein in the assembly and secretion of TGF-B 1

Kohei Miyazono, Anders Olofsson, Pascal Colosetti and Carl-Henrik Heldin Ludwig Institute for Cancer Research, Box 595, Biomedical Center, S-751 24 Uppsala, Sweden

Communicated by C.-H.Heldin

Transforming growth factor-,8 (TGF-/1) is synthesized as latent complexes with high molecular weights. The large latent complex of TGF-/1 in platelets is composed of three components, i.e. the mature TGF-/1, which is non-covalently associated with a disulphide-bonded complex of the N-terminal remnant of the TGF-/1 precursor (TGF-/1-latency associated peptide) and the latent TGF-/1 binding protein (LTBP). The TGF31-latency associated peptide is sufficient for the latency of TGF-/1, whereas the functions of LTBP remain to be elucidated. In a human erythroleukemia cell line, HEL, the production of the latent form of TGF-/31 was induced more than 100-fold by phorbol 12-myristate 13-acetate. Analysis by Northern blotting revealed that both the TGF-,B1 precursor and LTBP were induced in a coordinated fashion. Analysis by immunoprecipitation using antibodies against LTBP and the TGF-/1 precursor dimer revealed that LTBP has a molecular size of 205 kd under reducing conditions in this cell type, i.e. similar to that from cells transfected with cDNA for LTBP, but larger than the platelet form (125-160 kd). Limited tryptic digestion of LTBP in HEL cells and analysis by SDS-PAGE showed protein bands of similar sizes to those of platelet LTBP, suggesting that the difference in molecular sizes of LTBP involves cell-specific processing. The biosynthesis of the latent TGF-,B1 was studied by pulse - chase analysis. LTBP became covalently associated with the TGF-/31 precursor within 15 min after synthesis in this cell line. Secretion of the large latent TGF-/1 complex was observed as early as 30 min after the synthesis of LTBP; at the same time, a free form of LTBP not bound to the TGF-/31 precursor was seen. In contrast, the TGF-/1 precursor remained inside the cells in an unprocessed form for a longer time period and the TGF-/1 precursor dimer without LTBP was secreted only very slowly. Furthermore, the results of partial tryptic digestion of this molecule suggested that it contained improper disulphide bonding. These results suggest that LTBP plays a critical role in the assembly and secretion of the latent TGF-/1. Key words: binding protein/biosynthesis/latency/phorbol

ester/TGF-3

Introduction TGF-/s activities

family of proteins with multifunctional many different cell types (for reviews see

are a

on

Oxford University Press

Roberts and Sporn, 1990; Moses et al., 1990; Massague, 1990). TGF-/1 was initially identified as a growth factor that induced the growth of rodent fibroblasts in semi-solid agar. However, TGF-/s are now also known to be potent growth inhibitors for many different cell types, including epithelial cells, endothelial cells, hemqtopoietic progenitor cells and lymphocytes. In addition, TGF-3s potently induce the deposition of extracellular matrix proteins. So far, three different human isoforms of TGF-,3s have been reported, TGF-/1, -/2 and -/3. In addition, TGF-,B4 and -/5 have been identified in chicken and frog, respectively. Other proteins distantly related to TGF-/3 1 have also been described, such as inhibins/activins, Mullerian inhibitory substance and bone morphogenic proteins; many of these proteins have regulatory effects on cell growth and differentiation, but often show distinct effects from TGF-0s. TGF-j31 is secreted from the producer cells as latent complexes of high molecular weights. Since TGF-31 is a ubiquitous molecule, the activation of the latent complex is likely to be an important step in the action of this factor (Pircher et al., 1984; Wakefield et al., 1987; Lyons and Moses, 1990; Miyazono and Heldin, 1991). In human (Miyazono et al., 1988; Wakefield et al., 1988) and rat (Okada et al., 1989) platelets, the latent form of TGF-,B1 is comprised of three distinct components; mature TGF-/ 1 which is a disulphide-bonded dimer, the N-terminal remnant of the TGF-3 1 precursor and a novel type of protein denoted the latent TGF-3 1 -binding protein (LTBP). The N-terminal remnant of the TGF-/1 precursor forms a disulphide-bonded dimer, which is linked to a single molecule of LTBP by a disulphide bond. In human platelets, the mature form of TGF-/1 has been proteolytically cleaved off from the Nterminal remnant of the TGF-/1 precursor, but remains noncovalently associated with the rest of the complex (Miyazono et al., 1988; Wakefield et al., 1988). The Nterminal remnant of the TGF-/1 precursor is sufficient for TGF-/1 latency, since transfection of Chinese hamster ovary cells with TGF-/1 precursor cDNA yielded a latent TGF/31 complex without LTBP, which still was latent (Gentry et al., 1987). Therefore, the N-terminal remnant of the TGF/31 precursor has recently been denoted TGF-,31-latency associated peptide (/1-LAP) (Gentry and Nash, 1990). Furthermore, the latent TGF-/1 complex which includes LTBP is denoted the 'large latent complex', whereas the complex without LTBP is denoted the 'small latent complex' (Wakefield et al., 1989; Miyazono and Heldin, 1991). TGF/2 and -/3 have been also shown to be synthesized as latent complexes in Chinese hamster ovary cells (Brown et al., 1990). cDNA for LTBP was recently cloned from human foreskin fibroblasts (Kanzaki et al., 1990). The open reading frame of the cDNA sequences predicted a 1394 amino acid protein containing two types of cysteine-rich repeat sequences; 16 epidermal growth factor (EGF)-like repeats and three copies of a repeat sequence of a novel type. Thus, LTBP has a 1091

K.Miyazono et al.

unique structure, however, it is not necessary for the latency of TGF-(1 and its function in the complex is still unknown. In some of the EGF-like repeat sequences, (-hydroxylated asparagine residues were identified (Kanzaki et al., 1990), suggesting that LTBP is a Ca2+ binding protein (Dahlback et al., 1990). A molecule similar to human LTBP was also recently cloned from rat (Tsuji et al., 1990); this molecule is 336 amino acids longer in the N-terminus. The regulation of the biosynthesis of TGF-(s has recently been extensively studied and shown to be modulated by several different factors. Phorbol ester (Kim et al., 1989; Wager and Assoian, 1990), human T lymphotrophic virus type 1 Tax protein (Kim et al., 1990), and TGF-31 itself (Kim et al., 1989), induce the production of TGF-f1. The expression of the different isoforms of TGF-(s are regulated differently by TGF-(1 and -(2 (Bascom et al., 1989). In addition, production of TGF-,B2, but not TGF-(31, was shown to be upregulated in epidermal keratinocytes by retinoic acid (Glick et al., 1989). In order to understand the physiological roles of LTBP, it is important to elucidate the regulation of its expression in comparison with that of TGF(31 and the other isoforms of TGF-3. In this communication, we show that treatment of a human erythroleukemia cell line (HEL) with phorbol 12-myristate 13-acetate (PMA) leads to a coordinated induction of TGF(1 and LTBP. The assembly, secretion and processing of the components of the latent TGF-(31 have been studied. Our results suggest a role for LTBP in the proper assembly and secretion of the TGF-(31 molecule.

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Results Induction of latent TGF-/61 production by PMA HEL cells were incubated with various concentrations of PMA for 48 h in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cells were washed extensively and the media were changed to PMA-free RPMI 1640 medium with 1 mg/ml of bovine serum albumin. After an additional 48 h of incubation, cells were counted after trypsinization and the content of the latent TGF-(1 in the conditioned medium was investigated by a bioassay for TGFafter heat activation and by immunoblotting. By the treatment with PMA, the cells underwent a dramatic change in cell shape and a dose-dependent inhibition of HEL cell growth was observed (Figure IA). More than 50% decrease in the cell number was seen at 160 nM PMA; at this concentration of PMA, most of the cells, which without PMA grow in suspension, had attached to the culture dish. The growth inhibition of mink lung epithelial cells (CCL-64) was used as bioassay for TGF-(3. The conditioned media from HEL cells were activated by heating at 80°C for 10 min prior to assay (Brown et al., 1990). Incubation with PMA led to a dramatic dose-dependent increase in the production of TGF-(3 activity in HEL cells; at 1.6 /iM of PMA more than 100-fold increase in TGF-(3 production was found (Figure iB). That the growth inhibitory activity was due to TGF-(3 was verified by showing that it could be neutralized by an antibody against TGF-(3, J069 (R & D Systems) (not shown). The production of LTBP was investigated by immunoblotting using a rabbit antiserum (Ab 39) raised against LTBP purified from human platelets (Kanzaki et al., 1990). This antibody recognizes LTBP in a free form as well as in a complex with the (31-LAP dimer

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Fig. 1. Production of latent TGF-j31 by HEL cells after treatrnent with PMA. Cells (5 x 105/ml, 5 ml of media/culture) were treated with PMA for 48 h in RPMI 1640 in the presence of 10% fetal bovine serum. Then, the media were changed to PMA-free RPMI 1640 medium with 1 mg/ml of bovine serum albumin and cultured for an additional 48 h. (A) Cell number after treatment with various concentrations of PMA. (B) TGF-,B activity in the media from HEL cells measured by a growth inhibition assay using mink lung epithelial cells (CCL-64) after activation of TGF-f by heating at 80°C for 10 min. (C) Immunoblotting of HEL cell conditioned media under nonreducing conditions using an antibody against LTBP (Ab 39).

under nonreducing conditions. Analysis of the conditioned media from HEL cells under nonreducing conditions revealed an increase in LTBP seen as two components of 190 and 270 kd at higher concentrations of PMA (Figure IC). Thus, a coordinated induction of LTBP and TGF-(3 activity occurred as HEL cells differentiated in response to PMA. Further experiments were performed using HEL cells treated with 1.6 1t.M of PMA. The expression of mRNA after various time periods of PMA treatment in HEL cells was also investigated. LTBP transcripts of 7.0 and 5.2 kb and a TGF-(1 precursor

Role of the latent TGF-f1-binding protein

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Fig. 2. Northern blot analysis of HEL cells. RNA was prepared from HEL cells treated with 1.6 uM of PMA for different time periods. 20 jig of total RNA was subjected to Northern blotting and hybridized with probes for LTBP (A) and TGF-,B1 precursor (B). Hybridization of the filter with a probe against GAPDH confirmed that approximately equal amounts of RNA were loaded in each lane (not shown).

transcript of 2.5 kb was found to increase after PMA treatment and to reach a maximum after 48 h (Figure 2).

Transcripts for TGF-,B2 and TGF-f33 precursor were observed by Northern blotting (data not shown).

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Structure of the latent TGF481 in HEL cells It was previously shown that the recombinant LTBP produced in COS cells had a molecular mass of 190 kd under reducing conditions (Kanzaki et al., 1990), i.e. considerably larger than the platelet forms of LTBP, which are 125-160 kd after reduction (Miyazono et al., 1988). In order to characterize the structure of the latent TGF-( 1 in PMAtreated HEL cells, immunoprecipitation was performed after the cells had been incubated with PMA for 60 h, using Ab 39 raised against LTBP, as well as antibody LT-1, raised against the TGF-(B1 precursor purified from conditioned medium of Chinese hamster ovary cells transfected with TGF-l01 precursor cDNA. Analysis of Ab 39-immunoprecipitates from HEL cell medium under nonreducing conditions revealed that the free form of LTBP of 190 kd was seen, together with the 270 kd complex between LTBP and ,B1-LAP and the 23 kd TGF-,B1 molecule (Figure 3A). Since TGF-31 is noncovalently associated with the 270 kd complex, it coprecipitated by Ab 39 and was released in the presence of SDS (Miyazono et al., 1988). The amount of the 190 kd free form of LTBP varied between different experiments. After reduction, three specific bands of 205, 40 and 13 kd were seen, representing LTBP, the $1-LAP monomer and the TGF-(31 monomer, respectively. Antibody Ab 39 specifically recognizes LTBP; (31-LAP and TGF-(31 co-immunoprecipitated with LTBP. Immunoprecipitation of HEL cell medium by antibody LT-l gave essentially similar

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Fig. 3. Immunoprecipitation of latent TGF-,B1 in HEL cells treated with PMA. HEL cells were treated with 1.6 jM PMA for 60 h and labelled with [35S]cysteine and [35S]methionine for 2 h. Cells were washed and incubated with a 5-fold molar excess of unlabelled cysteine and methionine for 4 h. Conditioned media and cell lysates were collected and subjected to immunoprecipitation using Ab 39 and LT-1. Immunoprecipitates were analyzed by SDS-PAGE and fluorography in the absence or presence of dithiothreitol (DTT). For the competition of the binding to antibodies, 5 jig of purified LTBP, or 5 jg of purified TGF-,B1 precursor was added to Ab 39 and LT-1, respectively.

1093

K.Miyazono et al.

results. Antibody LT- 1 recognizes only the TGF-( 1 precursor, but LTBP was also co-immunoprecipitated in the complex. However, the nonreduced band of 190 kd observed after immunoprecipitation with Ab 39, was not detected, in support of the notion that it is a free form of LTBP (Figure 3A). Moreover, a 100 kd fuzzy band together with an 84 kd faint band was seen under nonreducing conditions, which may represent the small latent TGF-, 1 complex without LTBP (see below). Under reducing conditions, three specific bands of 205, 40 and 13 kd were seen, representing LTBP, monomeric ,B1-LAP and monomeric TGF-(1 respectively, as well as a faint 48 kd band, which has the expected size of a nonprocessed form of the TGF-f1 precursor. From the lysate of HEL cells, Ab 39 precipitated a component of a nonreduced size of 260 kd, which was reduced to components of 195 kd and 48 kd (Figure 3B). The 195 kd LTBP band is thus slightly smaller than the extracellular form, probably because of differences in glycosylation. Since the 48 kd protein was coprecipitated with Ab 39 and not observed under nonreducing conditions, it is probably an uncleaved precursor form of TGF-, 1, which forms a disulphide-bonded complex with LTBP (see further below). Under nonreducing conditions, immunoprecipitation by LT-1 yielded, in addition to the 260 kd large latent complex, an 84-90 kd doublet band (Figure 3B). Under reducing conditions, the 195 kd LTBP band was seen as well as a predominant 48 kd band, conforming to the notion that the 48 kd band represents an uncleaved TGF-3 1 precursor. The 84-90 kd doublet band observed under nonreducing conditions, might correspond to dimeric form of the 48 kd TGF-f31 precursor; it remains to be elucidated, however, why this component occurs as a doublet. Only very faint TGF-(1 bands were observed in the cell lysates, indicating that processing of TGF- 1 from (1-LAP occurs very inefficiently inside the cells. Enzymatic deglycosylation of the latent TGF-31 using endoglycosidase F/N-glycosidase F (Boehringer Mannheim GmbH) was performed using material immunoprecipitated by LT-1 (Figure 4). Endoglycosidase F/N-glycosidase F treatment, which hydrolyzes the complex- and the high mannose-types of N-linked carbohydrate structures, induced a decrease in the size of LTBP by less than 10 kd, whereas in the conditioned media the 48 kd TGF-( 1 precursor shifted to 43 kd and $1-LAP from 40 kd to 31 kd. Similarly, in the cell lysates, the 48 kd band shifted to 45 and 43 kd. These data are consistent with the presence of three potential acceptor sites for N-linked glycosylation in the primary sequence of TGF-(1 (Derynck et al., 1985), all of which are localized in the 3 1-LAP portion. There are seven potential N-glycosylation sites in LTBP (Kanzaki et al., 1990). The shift in the molecular size after treatment with endoglycosidase F/N-glycosidase F of less than 10 kd is less than expected, if all potential N-glycosylation sites were used. Since the calculated molecular weight of the primary translated product of LTBP without signal sequence is only 150 kd, it is possible that some of the N-glycosylation sites were not cleaved by endoglycosidase F/N-glycosidase F under the conditions used, or alternatively that additional post-translational modifications occur in LTBP, e.g. 0-linked glycosylation. It is also possible that the deglycosylated 195 kd LTBP molecule represents a larger variant of the molecule, e.g. corresponding to that recently identified in the rat (Tsuji et al., 1990). -

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Fig. 4. Enzymatic deglycosylation of the latent TGF-fl1. HEL cells were metabolically labelled as described in the legend of Figure 3. The conditioned media and the cell lysates were collected and subjected to immunoprecipitation using antibody LT-1. The samples were then treated with 0.2 U of endoglycosidase F/N-glycosidase F (Boehringer Mannheim GmbH) at 37°C for 16 h in a buffer containing 100 mM phosphate, pH 6.1, 50 mM EDTA, 1% Triton X-100, 0.1% SDS and 0.1% 3mercaptoethanol. Samples were alkylated by iodoacetamide and then analyzed by SDS-PAGE and fluorography.

Tryptic digestion of the latent TGF-,B 1 In order to investigate the possibility that the difference in the molecular sizes of LTBP in human platelets and HEL cells is due to proteolysis, the conditioned media of metabolically labelled HEL cells were immunoprecipitated with Ab 39 and then treated with trypsin for different time periods. Analysis of the samples by SDS-PAGE under reducing conditions revealed that already after 2 min of incubation with trypsin, the 205 kd LTBP shifted to bands of 125-160 kd (Figure SA), i.e. similar to the platelet forms of LTBP (Miyazono et al., 1988). Under nonreducing conditions, the 270 kd complex between LTBP and the (1-LAP dimer shifted to 210 kd (Figure SB), also very similar to the platelet form. These results suggest that the difference in molecular mass of LTBP in HEL cells and human platelets is due to cell-specific proteolysis. In order to explore the possibility that the 48 kd molecule recognized by LT-1 antibody in the cell lysates of metabolically labelled HEL cells represents an uncleaved TGF-(1 precursor, LT-1 immunoprecipitates were subjected to tryptic digestion (Figure 6). The 48 kd band was found to be rapidly cleaved to a 36-34 kd doublet and concomitantly a 13 kd band increased in strength (Figure 6A). In addition, bands of 10 and 11 kd were seen, probably representing further degradation products. Under nonreducing conditions, the 84-90 kd doublet bands were

Role of the latent TGF-f1 -binding protein

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Fig. 5. Tryptic digestion of LTBP. HEL cells were labelled by [35S]cysteine and [35S]methionine for 2 h and then incubated with a 5-fold molar of unlabelled cysteine and methionine for 4 h. Conditioned medium was immunoprecipitated with Ab 39. Samples were then treated with 0.5 jig of trypsin in 50 /d of 50 mM Tris-HCI, pH 7.4, 5 mM CaCl2 at 37°C for various time periods and quenched by the addition of 2 tzg of soybean trypsin inhibitor. Samples were analyzed by SDS-PAGE in the presence (A) or absence (B) of DTT. excess

partially converted to a component of 64 kd, together with the appearance of a 23 kd band (Figure 6B). These results suggest that the 48 kd component (84-90 kd without reduction) represents an uncleaved form of the TGF-,13 precursor and that trypsin causes a clip at the dibasic amino acid residue site which is used during the normal processing of TGF-1 I (Derynck et al., 1985), yielding the 13 kd TGF131 monomer (23 kd without reduction), and the 36-34 kd 11-LAP (64 kd without reduction). It is noteworthy that in the TGF-11 precursor expressed in Chinese hamster ovary cells, a disulphide linkage between ,31-LAP and mature

TGF-31 was seen (Gentry et al., 1988; Brunner et al., 1989). A substantial part of the 84-90 kd TGF-131 precursor remained at this size when analyzed under nonreducing conditions after trypsin treatment (Figure 6B), despite the fact that all 48 kd molecules were cleaved (Figure 6A). This suggests that at least a part of the TGF-11 precursor produced by HEL cells also has anomalous disulphide bonding between TGF-1 1 and $13-LAP. Assembly, secretion and processing of the latent TGF-,3 1 in HEL cells In order to characterize the assembly and secretion of the components of the latent TGF-11 in HEL cells, a pulse chase analysis was performed. HEL cells treated with 1.6 zIM of PMA for 60 h were labelled with [35S]cysteine and [35S]methionine for 15 min and then chased for various time periods (Figure 7). Analysis of HEL cell lysates by Ab 39 (Figure 7A), revealed that after the synthesis, LTBP

bound to the TGF-1 1 precursor within 15 min. This conclusion is based on the observation that a 260 kd complex between LTBP and the TGF-1 1 precursor was observed under nonreducing conditions. In the conditioned media (Figure 7B), LTBP was seen after 30 min of chase, as components of 270 and 190 kd, suggesting that the secretion of the large latent TGF-13 1 complex and the free form of LTBP started at the same time. Studies using the LT- 1 antibody gave additional information. LTBP co-immunoprecipitated with the TGF131 precursor as early as 15 min after synthesis and was observed as a 260 kd band without reduction and a 195 kd band with reduction (Figure 7C). Thus, LTBP is assembled with the TGF-131 precursor early after biosynthesis. A 44 kd band was observed under nonreducing conditions, probably representing a monomeric TGF-1 1 precursor, migrating slightly faster than its reduced 48 kd counterpart. Dimerized TGF-1 1 precursor (84-90 kd) was then observed from 15 min after synthesis. The large latent TGF-131 complex was the major form secreted from the producer cells (Figure 7D). Under nonreducing conditions, the 84 kd band representing the dimeric form of the TGF-13 1 precursor was seen after 120 min of chase. Thus, some of the TGF-1 1 precursor dimer without LTBP was secreted, but only relatively slowly compared with the large latent TGF-13 complex. In order to study the secretion and processing of the latent TGF-11 in detail, cells were labelled with [35S]cysteine and [35S]methionine for 1 h and then chased for various time 1095

K.Miyazono et al.

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Since blood platelets originate from bone marrow megakaryocytes, the latent form of TGF-fll present in human platelets might have been produced in this cell type. In porcine platelets, both TGF-f1 and TGF-j2 homodimers as well as the heterodimer have been observed (Cheifetz et

1098

3

and

al., 1987), whereas in human platelets, the major form of TGF-j is TGF-f 1 (Cheifetz et al., 1987; Miyazono et al., 1988). Northern blot analysis revealed that the major form of TGF-j in the PMA-treated HEL cells is TGF-j1 (Figure 2). Preliminary studies by immunoprecipitation using

Role of the latent TGF-31 -binding protein

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antibodies to synthetic peptides corresponding to the TGF/1, -/2, and -33 precursors, also disclosed that the PMAtreated HEL cells produced only TGF-31 (K.Miyazono, unpublished data). The large latent TGF-31 complex contains two different gene products, i.e. the TGF-f31 precursor and LTBP. In this study, we have shown that both TGF-31 and LTBP are induced in HEL cells after treatment with PMA. The coordinated expression of TGF-/1 and LTBP indicate that the production of these proteins may be regulated by the same pathways. TGF-32 and -33 have also been shown to be synthesized as latent forms (Brown et al., 1990), however, detailed knowledge about the structures of these complexes is lacking. It was shown that the promotor regions of the genes for TGF-f2 (Malipiero et al., 1990; Noma et al., 1991) and -,B3 (Lafyatis et al., 1990) are considerably different compared with that of the TGF-f 1 gene. In order to understand the regulated expression of LTBP, it will be important to characterize the promotor region of the LTBP gene and to compare it with those of the genes of the different isoforms of TGF-/. It was recently shown that the induction of TGF-/1 by phorbol ester in a monocytic leukemia cell line U937, was due to post-transcriptional stabilization of the mRNA (Wager and Assoian, 1990); it will be interesting to investigate whether a similar mechanism operates also in HEL cells and whether stabilization of LTBP mRNA also occurs in response to PMA treatment. Recently, Tsuji et al. (1990) reported the cDNA cloning of LTBP from a rat megakaryocyte library (referred as the large subunit of the TGF-/ masking protein). The open reading frame of the cDNA codes for 1712 amino acids, i.e. 336 amino acids longer than the cDNA clone obtained from human foreskin fibroblasts (Kanzaki et al., 1990). The sequences differ in the N-terminal part; the possibility that the presence of two different forms of LTBP is related to the observation of two different mRNA spieces (Figure 2), is currently being explored.

1

in HEL cells. For discussion,

4

Free form of LTBP see

text.

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LTBP isolated from human platelets has lower molecular weights compared with that from HEL cells or from COS cells transfected with LTBP cDNA (Kanzaki et al., 1990). This is probably due to different proteolytic processing of a single protein, since LTBP derived from HEL cells was converted to a family of components of similar sizes to those in platelets after limited proteolysis by trypsin (Figure 5). LTBP is made up of two different types of cysteine-rich repeat sequences, i.e. EGF-like repeat sequences and repeats of a novel type with eight cysteine residues (Kanzaki et al., 1990). EGF-like repeat sequences are observed in many different proteins, including growth factors, cell surface receptors, blood coagulation factors, cell adhesion molecules and proteins with presumed functions in developmental processes (Davis, 1990). The functions of the EGF-like repeat sequences in LTBP remain to be elucidated. The processing of the TGF-/ 1 precursor occurs relatively slowly. Inside HEL cells, bands corresponding to the mature form of TGF-/31 could not be observed. Instead, the 48 kd precursor protein could be precipitated by both Ab 39 and LT-l (Figure 8A and C). In the conditioned media, the 48 kd band was also visible, but the 40 kd /1-LAP band and 13 kd TGF-/B1 band were predominant forms (Figure 8 B and D). These data indicate that the processing occurs in conjunction with, or just after, secretion. The TGF-/1 precursor dimer, which failed to bind to LTBP, was secreted only slowly, which is consistent with the observation made by Sha et al. (1989) that recombinant TGF-/31 precursor is secreted slowly, with only 50% secreted after 6 h. In addition, recombinant TGF-/1 has been found to be produced as a latent complex, part of which has an anomalous structure with a disulphide bond between /31-LAP and mature TGF-/31 (Gentry et al., 1988; Brunner et al., 1989). Mature TGF-/1 could not be released from the anomalous complex even after treatment with acid, heating or by the addition of SDS. In contrast, we have not observed any disulphide linkage between /31-LAP and TGF1099

K.Miyazono et al.

031 in the latent TGF-1 I containing LTBP purified from human platelets (Miyazono et al., 1988). These results, together with the observation of the present study that a part

of the TGF-,1 precursor dimer which lacks LTBP, has a disulphide linkage between $13-LAP and the mature TGF131 (Figure 6 and 8D), suggest that the binding of LTBP to

the TGF-1 1 precursor may prevent the anomalous disulphide

bonding and allow the production of proper TGF-131. The results obtained on the biosynthesis and processing of latent TGF-1 1 in HEL cells are schematically illustrated in Figure 9. The TGF-1 I precursor and LTBP are synthesized as distinct gene products, both of which have signal sequences and enter the secretory pathway. The TGF131 precursor undergoes dimerization and some of the molecules become associated with a single molecule of LTBP. This large latent complex is efficiently secreted from the producer cells. The TGF-31 precursor is proteolytically cleaved to yield the mature TGF-j31 molecule in conjunction, or just after, secretion, but TGF-3 1 remains noncovalently associated with the complex. Some LTBP is also secreted as a free form, not bound to the TGF-31 precursor. LTBP may, at least in some cell types, undergo proteolytic processing in the N- or C-terminals. The TGF-11 precursor also occurs as small latent complexes not containing LTBP. Part of such complexes has anomalous disulphide bonding. The small latent TGF-fl1 complexes are secreted and processed only very slowly. In conclusion, we have shown that LTBP associates with the TGF-j1 precursor rapidly inside the cells and that this association is important for the proper assembly and secretion

of TGF-j1. Whether LTBP has additional functions in the latent complex remains to be determined.

Materials and methods Assay for TGF-,/ activity TGF-3 activity was monitored as inhibition of the growth of mink lung epithelial cells, CCL-64 (American Tissue Culture Collection), using a [3H]thymidine incorporation assay (Cone et al., 1988) with a slight

modification. CCL-64 cells were transferred to 24-well tissue culture plates in Dulbecco's modified Eagle's medium containing 1 % fetal bovine serum and antibiotics. After 24 h of incubation, the medium was changed and the test samples were added. After an additional 20 h of incubation, cells were pulsed with 0.2 14Ci of [3H]thymidine (6.7 Ci/mmol, 1 mCi = 37 MBq, New England Nuclear) for 2 h. The 3H radioactivity incorporated into DNA was determined as previously described (Miyazono et al., 1987). Northern blot hybridization HEL cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and antibiotics. HEL cells in roller bottles were treated with 1.6 zM of PMA (Sigma) for various time periods. Total RNA was prepared by disruption of the cells in 4 M guanidine isothiocyanate and centrifugation through a cesium chloride cushion (Chirgwin et al., 1979). After phenol:chloroform (1:1) extraction and ethanol precipitation, 20 ,ug aliquots of total RNA were electrophoresed in a 0.7% agarose gel in the presence of formaldehyde and blotted to nitrocellulose filters. The blots were hybridized at 40°C for 16 h in 45% formamide, 6 xSSPE (1 xSSPE = 150 mM NaCl, 10 mM sodium phosphate, pH 7.4, 1 mM EDTA), to xDenhardt's solution, 0.1% SDS and 0.1 mg/ml salmon sperm DNA (Claesson-Welsh et al., 1989). 32P-labelled probes corresponding to nucleotides 1675 -2150 of the LTBP cDNA (Kanzaki et al., 1990), or an oligonucleotide probe of TGF-f31 corresponding to nucleotides 1184-1264 (Derynck et al., 1985; Derynck et al., 1987), were added at 2 x 106 c.p.m./ml. The filters were washed 2x 15 min in 2xSSC (1 xSSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0) and 0.1 % SDS at 220C and then once in 0.5 xSSC and 0.1% SDS for 15 min at 55°C. The filters were stripped by 0.01 xSSC, 0.1% SDS at 70°C for 2 h and hybridized with 32P-labelled probe of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2 x 105 c.p.m./ml) as a control.

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Antibodies The rabbit polyclonal antibody Ab 39 against purified LTBP (Miyazono et al., 1988) was made according to the procedure as described (Miyazono and Heldin, 1989). The rabbit antibody LT-1 was raised against purified recombinant TGF-,1 precursor, which was obtained from Chinese hamster ovary cells transfected with TGF-,B1 cDNA (gift from Hideya Ohashi and Kiyoshi Miyagawa, Tokyo, Japan). Purification was performed by serial chromatography steps, using S-Sepharose, Alkyl-Superose and Superose 6 columns (Pharmacia) (K.Miyazono, unpublished data). A neutralizing antibody against TGF-fl1 (J069) was purchased from R & D Systems. Immunoblot analysis Conditioned media from HEL cells were concentrated using Minicon concentrator (Amicon). Samples were subjected to SDS-PAGE in 7.5% polyacrylamide gels and transferred to nitrocellulose membranes in a buffer containing 20% methanol, 192 mM glycine and 25 mM Tris-HCI, pH 8.4 at 100 V. The nitrocellulose membranes were blocked by incubation in Tris-buffered saline (Tris-HCI, pH 7.4, 150 mM NaCI) containing 3% bovine serum albumin and then incubated with antibodies (1:50 dilution) in the same buffer for 16 h. The nitrocellulose membranes were then washed, incubated with 125I-labelled staphylococcal protein A (3 x 105 c.p.m./ml), washed again, and finally subjected to autoradiography as described (Miyazono and Heldin, 1989). Metabolic labellinq and immunoprecipitation HEL cells in 75 cm or 25 cm2 flasks were treated with 1.6 ,IM of PMA for 60 h. Metabolic labelling and immunoprecipitation were performed as previously described (Usuki et al., 1989), with a slight modification. Briefly, the cells were washed three times and given cysteine- and methionine-free Dulbecco's modified Eagle's medium supplemented with 20 mM HEPES, pH 7.4 and 1 mg/ml of bovine serum albumin. The cells then received [35S]cysteine (0.2 mCi/ml) and [35S]methionine (0.2 mCi/mI), and were incubated at 37°C for various time periods. After incubation, the cells were washed and incubated in a medium containing a 5-fold molar excess of unlabelled cysteine and methionine for various time periods. The conditioned media were collected; the cells were solubilized for 20 min on ice in lysis buffer containing 0.15 M NaCl, 50 mM Tris-HCI, pH 7.4, 1% Triton X-100, 1% deoxycholate, and 150 kallikrein inhibitor units of aprotinin per ml. The conditioned media and cell lysates were incubated with 5 td/ml of preimmune serum for 2 h at 4°C and given 80 pI/ml of protein A-Sepharose (Pharmacia) slurry (50% packed beads in 0.15 M NaCl, 20 mM Tris-HCI, pH 7.4, 0.2% Triton X-100). The mixture was incubated for an additional 45 min at 4°C with gentle mixing. The beads were spun down by centrifugation and the supematants were collected. The same procedures were repeated twice for the conditioned media and three times for the cell lysates. Samples were then incubated with 5 td/ml of rabbit antisera for 2 h at 4°C; 80 ,ul/ml of protein A-Sepharose slurry was then added and incubation prolonged for 45 min at 4°C. The beads were collected after centrifugation, washed three times with lysis buffer, twice with 0.5 M NaCl, 20 mM Tris-HCI, pH 7.4, 0.2 % Triton XI00 and once with 20 mM Tris-HCI, pH 7.4. In some experiments, the samples were subjected to digestion with endoglycosidase F/N-glycosidase F or trypsin. Then, the proteins bound to the beads were eluted by addition of 50 tll of SDS sample buffer (Blobel and Dobberstein, 1975), and heated for 3 min at 95°C. Samples were then analyzed by SDS-PAGE using 5-18% polyacrylamide gradient gels (Blobel and Dobberstein, 1975). The gels were soaked in Amplify (Amersham) for 30 min, dried and subjected to fluorography.

Acknowledgements We thank Hideya Ohashi and Kiyoshi Miyagawa for providing recombinant TGF-,B1 precursor, Keiko Yuki, Anita Moren and Susanne Grimsby for technical assistance and Lena Claesson-Welsh for discussion.

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