©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation of a Novel Latent Transforming Growth Factor- Binding Protein Gene (LTBP-3) (*)

Wushan Yin , Elizabeth Smiley , John Germiller , Robert P. Mecham (1), Jane B. Florer (2), Richard J. Wenstrup (2), Jeffrey Bonadio (§)

From the (1) Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109-0650, the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, and the (2) Division of Human Genetics, Department of Pediatrics, Children's Hospital Research Foundation, Cincinnati, Ohio 45229-3039

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

This paper reports the molecular cloning of a novel gene in the mouse that shows structural similarities to the microfibril protein fibrillin and to the latent transforming growth factor- (TGF-) binding protein (LTBP), a component of the latent TGF- complex. The gene was initially isolated during a low stringency polymerase chain reaction screen of a NIH 3T3 cell cDNA library using primers that amplify a human fibrillin-1 epidermal growth factor-like repeat. Three lines of evidence suggest that the mouse gene is a third member of the LTBP gene family, which we designate LTBP-3. First, the deduced polypeptide, which consists of 15 epidermal growth factor-like repeats, 3 TGF binding protein repeats, and 2 proline- and glycine-rich sequences, shows 38.4% identity with LTBP-1 but only 27% identity with fibrillin-1. Second, the gene appears to be co-expressed in developing mouse tissues with TGF-. Third, immunoprecipitation studies using mouse preosteoblast MC3T3-E1 cells and a specific anti-peptide polyclonal antiserum reveal that the mouse polypeptide forms a complex with the TGF-1 precursor. Finally, we note that the LTBP-3 gene was recently localized to a distinct genetic locus (Li, X., Yin, W., Perez-Jurado, L., Bonadio, J., and Francke, U. (1995) Mamm. Genome 6, 42-45). Identification of a third binding protein provides further insight into a mechanism by which latent TGF- complexes can be targeted to connective tissue matrices and cells.


INTRODUCTION

Transforming growth factor- (TGF-s)() represents a family of structurally related molecules with diverse effects on mammalian cell shape, growth, and differentiation (Roberts and Sporn, 1990). Initially synthesized as a precursor consisting of an amino-terminal propeptide followed by mature TGF-, two chains of nascent pro-TGF- associate in most tissues to form a M 106,000 inactive disulfide-bonded dimer. Homodimers are most common, but heterodimers have also been described (Cheifetz et al., 1987; Ogawa et al., 1992). During biosynthesis, the mature TGF- dimer is cleaved from the propeptide dimer. TGF- latency results in part from the noncovalent association of propeptide and mature TGF- dimers (Pircher et al. (1984, 1986), Wakefield et al. (1987), Millan et al. (1991), and see also Miyazono and Heldin (1989)). Consequently, the propeptide dimer is often referred to as the latency-associated protein, and this protein plus the disulfide-bonded TGF- dimer are also known as the small latent complex. In the extracellular space, small latent complexes must be dissociated to activate mature TGF-. The mechanism of activation of the latent complex is thought to be one of the most important steps governing TGF- effects (Lyons et al., 1988; Antonelli-Orlidge et al., 1989; Twardzik et al., 1990; Sato et al., 1993).

In certain lines of cultured cells, small latent growth factor complexes may contain additional high molecular weight proteins. The best characterized of these high molecular weight proteins is the latent TGF- binding protein, or LTBP (Miyazono et al., 1988; Kanzaki et al., 1990; Tsuji et al., 1990; Olofsson et al., 1992; Taketazu et al., 1994). LTBP produced by different cell types is heterogeneous in size, perhaps because of alternative splicing or because of tissue-specific proteolytic processing (Miyazono et al., 1988; Wakefield et al., 1988; Kanzaki et al., 1990; Tsuji et al., 1990). Latent TGF- complexes that contain LTBP are known as large latent complexes. LTBP has no known covalent linkage to mature TGF-, but rather it is linked by a disulfide bond to the latency-associated protein.

Two LTBPs have been isolated to date. The deduced human LTBP-1 amino acid sequence is composed of a signal peptide, 16 epidermal growth factor-like repeats with the potential to bind calcium (EGF-CB repeats), two copies of a unique motif containing 8 cysteine residues, an RGD cell attachment motif, and an 8-amino acid motif identical to the cell binding domain of the laminin B2 chain (AGPHCEKC) (Kanzaki et al., 1990). There is evidence that LTBP-1 binds calcium, which, in turn, induces a structural change that protects LTBP from proteolytic attack (Colosetti et al., 1993). LTBP-2 shows 41% sequence identity to LTBP-1, and its structural domains show a similar overall organization (Moren et al., 1994).

While the functions of LTBP-1 and LTBP-2 presently are unknown, several ideas have been put forward in the literature. First, LTBP may regulate the intracellular biosynthesis of latent TGF- precursors. Cultured erythroleukemia cells efficiently assemble and secrete large latent TGF- complexes, whereas they slowly secrete small latent TGF- complexes that contain anomalous disulfide bonds (Miyazono et al., 1991, 1992). Therefore, LTBP may facilitate the normal assembly and secretion of latent TGF- complexes. Second, LTBP may target latent TGF- to specific types of connective tissue. Recent evidence suggests that the large latent TGF- complex is covalently bound to the extracellular matrix via LTBP (Taipale et al., 1994). Based on these observations, LTBP has been referred to as a ``matrix receptor'', i.e. a secreted protein that targets and stores latent growth factors such as TGF- to the extracellular matrix. Third, LTBP may modulate the activation of latent complexes. This idea is based in part on recent evidence that suggests that mature TGF- is released from extracellular storage sites by proteases such as plasmin and thrombin and that LTBP may protect small latent complexes from proteolytic attack (Falcone et al., 1993; Benezra et al., 1993; Taipale et al., 1994), i.e. protease activity may govern the effect of TGF- in tissues, but LTBP may modulate this activity. Fourth, LTBP may play an important role in targeting the latent TGF- complex to the cell surface, allowing latent TGF- to be efficiently activated (Flaumenhaft et al., 1993).

In this paper, we report the molecular cloning of a new gene in the mouse that is related to LTBP-1 and LTBP-2. This conclusion is supported by (i) comparative analysis of DNA sequence identity, (ii) an evaluation of gene expression in developing tissues, and (iii) immunoprecipitation studies that show that that new mouse gene product forms specific complex with TGF- 1. A recent study has also shown that the new mouse gene is localized to a distinct genetic locus (Li et al., 1994).Taken together, these results suggest that LTBP is a gene family of at least three members and that an appropriate designation for the new gene is LTBP-3. Identification of a third binding protein family member provides further insight into the mechanism by which latent TGF- complexes can be targeted to connective tissue matrices and cells.


EXPERIMENTAL PROCEDURES

cDNA Cloning

Aliquots (typically 40-50,000 plaque-forming units) of phage particles from a cDNA library in the ZAP II vector made from NIH 3T3 cell mRNA (Stratagene) and fresh overnight XL1-Blue cells (grown in Luria broth supplemented with 0.4% maltose in 10 mM MgSO) were mixed, incubated for 15 min at 37 °C, mixed again with 9 ml of liquid (50 °C) top layer agarose (NZY broth plus 0.75% agarose), and then spread evenly onto freshly poured 150-mm NZY-agar plates. Standard methods were used for the preparation of plaque-lifts and filter hybridization (42 °C, in buffer containing 50% formamide, 5 SSPE, 1 Denhardt's solution, 0.1% SDS, 100 mg/ml salmon sperm DNA, 100 mg/ml heparin). Filters were washed progressively to high stringency (0.1 SSC, 0.1% SDS, 65 °C). cDNA probes were radiolabeled by the nick translation method using commercially available reagents and protocols (Sure Site kit, Novagen). Purified phage clones were converted to pBluescript plasmid clones, which were sequenced using Sequenase (version 2.0) as described previously (Chen et al., 1993). ()Sequence alignment and identity was determined using sequence analysis programs from the Genetics Computer Group (MacVector).

Tissue In Situ Hybridization

To prepare normal sense and antisense probes, a unique 342-base pair fragment from the 3`-untranslated region (+3973 to +4314, counting the A of the initiator Met codon as +1; see Fig. 1, ish) was subcloned into the pBluescript KS+ plasmid (Stratagene, Inc.). Template DNA was linearized with either EcoRI or BamHI, extracted, and precipitated with ethanol. Sense and antisense transcripts were generated from 1 µg of template with T3 and T7 polymerases in the presence of [S]UTP at >6 mCi/ml (Amersham Corp., >1200 Ci/mmol) and 1.6 units/ml RNasin (Promega), with the remaining in vitro transcription reagents provided in a kit (SureSite, Novagen Inc.). After transcription at 37 °C for 1 h, DNA templates were removed by a 15-min digest at 37 °C with 0.5 units/ml RNase-free DNase I, extracted and precipitated with ethanol. Riboprobes were hydrolyzed to an average final length of 150 base pairs by incubating in 40 mM NaHCO, 60 mM NaCO, 80 mM dithiothreitol for 40 min at 60 °C. Hydrolysis was terminated by the addition of sodium acetate, pH 6.0, and glacial acetic acid to 0.09 M and 0.56% (v/v), respectively, and the probes were then ethanol-precipitated, dissolved in 0.1 M dithiothreitol, counted, and stored at 20 °C until use. Day 8.5-9.0, day 13.5, and day 16.5 mouse embryo tissue sections (Novagen) and the in situ hybridization protocol were exactly as described (Chen et al., 1993).


Figure 1: Overlapping mouse cDNA clones representing the mouse LTBP-3 gene sequence. A partial representation of restriction sites is shown. N, NcoI; P, PvuII; R, RsaII; B, BamHI; H, HindIII. The numbering system at the margin of each cDNA fragment and at the bottom of the figure assumes that the A of the initiator Met codon is nucleotide 1.



Northern Analysis

MC3T3-E1 cell poly(A) RNA (2-10-µg aliquots) was electrophoresed on a 1.25% agarose, 2.2 M formaldehyde gel and then transferred to a nylon membrane (Hybond-N, Amersham Corp.). The RNA was cross-linked to the membrane by exposure to a UV light source (1.2 10mJ/cm, UV Stratalinker 2400, Stratagene) and then prehybridized for >15 min at 65 °C in Rapid-Hyb buffer (Amersham, Corp.). A specific cDNA probe consisting solely of untranslated sequence from the 3` end of the transcript was P-labeled by random priming and used for hybridization (2 h at 65 °C). Blots were washed progressively to high stringency (0.1 SSC, 0.1% SDS, 65 °C), and then placed against x-ray film with intensifying screens (XAR, Kodak) at 86 °C.

Antibody Preparation

LTBP-3 antibodies were raised against a unique peptide sequence found in domain 2 (amino acids 155-167). Peptide 274 (GESVASKHAIYAVC) was synthesized using an ABI model 431A synthesizer employing FastMoc chemistry. The sequence was confirmed using an ABI 473 protein sequencer. A cysteine residue was added to the carboxyl terminus to facilitate cross-linking to carrier proteins. For antibody production, the synthetic peptide was coupled to rabbit serum albumin using m-maleimidobenzoic acid N-hydroxysuccinimide ester at a substitution of 7.5 mg of peptide/mg of rabbit serum albumin. One mg of the peptide-rabbit serum albumin conjugate in 1 ml of Freund's complete adjuvant was injected subcutaneously at 10 different sites along the backs of rabbits. Beginning at 3 weeks after initial immunization, the rabbits were given biweekly booster injections of 1 mg of peptide-rabbit serum albumin in 100 µl of Freund's incomplete adjuvant. IgG was prepared by mixing immune serum with caprylic acid (0.7 ml caprylic acid/ml of serum), stirring for 30 min, and centrifuging at 5000 g for 10 min. The supernatant was decanted and dialyzed against two changes of phosphate-buffered saline (PBS) overnight at 4 °C. The antibody solution was then affinity purified by passing it over a column containing the immunizing peptide coupled to Affi-Gel 10 affinity support. Bound antibodies were eluted with 0.2 M glycine, pH 2.3, immediately dialyzed against PBS, and concentrated to 1 mg/ml prior to storage at 70 °C.

Transfection

Transient transfection was performed using standard protocols (Sambrook et al., 1989). Briefly, subconfluent cells (covering 20% of a 100-mm plastic tissue culture dish) were washed 2 times in Dulbecco's modified Eagle's medium tissue culture medium (Life Technologies, Inc.) and then incubated for 3 h at 37 °C in a sterile mixture of DEAE-dextran (0.25 mg/ml), chloroquine (55 mg/ml), and 15 µg of plasmid DNA (Courey and Tjian, 1988). Cells then were shocked by incubation with 10% dimethyl sulfoxide in sterile PBS for 2 min at 37 °C, washed 2 with Dulbecco's modified Eagle's medium (Sambrook et al., 1989), and incubated in Dulbecco's modified Eagle's medium plus 10% fetal calf serum and antibiotics for 72 h at 37 °C.

Immunoprecipitation

For immunoprecipitation, 1 ml of antibody (1:400 final concentration, in PBS-TDS buffer (0.38 mM NaCl, 2.7 mM KCl, 8.1 mM NaHPO, 1.5 mM KHPO, 1% Triton X-100, 0.5% deoxycholic acid, and 0.1% SDS) was added to 1 ml of radiolabeled medium proteins. The mixture was incubated with shaking at 4 °C for 1 h, protein A-Sepharose CL-4B beads were added (200 µl, 10% suspension), and this mixture was incubated with shaking for 1 additional h at 4 °C. Immunoprecipitated proteins were pelleted by brief centrifugation, the pellet was washed 6 times with PBS-TDS buffer, 2 protein loading dye was added, and the samples were boiled for 5 min and then fractionated on 4-18% gradient SDS-polyacrylamide gel electrophoresis (Bonadio et al., 1985). Cold molecular weight markers (200-14.3 kDa, Rainbow mix, Amersham Corp.) were used to estimate molecular weight. The gel was dried and exposed to film for the indicated time at room temperature.

Western Analysis

Fractionated proteins within SDS-polyacrylamide gels were transferred to a nitrocellulose filter for 2 h using Tris-glycine-methanol buffer, pH 8.3, at 0.5 mA/cm. The filter was blocked, incubated with nonfat milk plus antibody (1:1000 dilution) for 2 h, and washed. Antibody staining was visualized using the ECL Western blotting reagent (Amersham Corp.) according to the manufacturer's protocols.


RESULTS

Molecular Cloning of a Mouse Gene Related to Human Fibrillin-1 and to LTBP

While cloning mouse fibrillin-1 (Yin et al., 1995), a 3T3 cell cDNA library was PCR amplified using human fibrillin-1 oligonucleotide primers. A PCR fragment with low homology to human fibrillin-1 was obtained. This result was surprising because the human and mouse fibrillin-1 genes share >95% coding sequence identity. To investigate the possibility that the low homology mouse PCR fragment was derived from a related (fibrillin-like) cDNA, the 3T3 cell cDNA library was screened at high stringency using the PCR fragment as the probe. A walking strategy eventually yielded a series of overlapping cDNA clones (Fig. 1).

Sequence analysis of cDNA clones revealed an open reading frame of 3,753 nucleotides. A methionine codon in a favorable context for translation initiation was provisionally designated the translation start site (CCTGCGATGC; see Kozak (1991)). The initiator methionine was followed by a characteristic signal sequence of 21 amino acids. Beyond this, the conceptual amino acid sequence appeared to be organized into five structurally distinct domains (Fig. 2). Domain 1 was a 28-amino acid segment (amino acids 22-49) with a net basic charge (estimated pI, 12.36). Domain 2 (amino acids 50-439) consisted of an EGF-like repeat, a 135-amino acid segment that was proline-rich (20.7%) and glycine-rich (11.8%) but not cysteine-rich, a Fib-motif (Pereira et al., 1993), an EGF-CB repeat, and a TGF-bp repeat. Domain 3 (amino acids 440-552) was characterized by its high proline content (21%). Domain 4 (amino acids 553-1229) consisted of 14 consecutive cysteine-rich repeats. Based on structural homologies, 12/14 repeats were EGF-CB motifs (Handford et al., 1990), whereas 2/14 were TGF-bp motifs (Kanzaki et al., 1990). Domain 5 was a unique 22-amino acid segment at the carboxyl terminus (amino acids 1230-1251).


Figure 2:A, schematic showing the structure of mouse fibrillin-1 ( top), the LTBP-3 gene product ( middle), and human LTBP-1 ( bottom). Structural domains are denoted below each diagram. Note that regions A-E of mouse fibrillin-1 (Yin et al., 1995) correspond to domains 1-5 of the mouse LTBP-3 gene product and LTBP. Symbols designate the following structural elements: EGF-CB repeats, open rectangles; TGF-bp repeats, open ovals; Fib motif, open circle; TGF-bp-like repeat, patterned oval; cysteine-rich sequences, patterned rectangles; proline/glycine-rich region, thin curved line, domain 2; proline-rich region, thick curved line, domain #3. Symbols designating the signal peptide have been deleted for simplicity. B, cDNA and deduced amino acid sequence of the mouse LTBP-2 gene. The boundaries of domains 1-5 are denoted by upward pointing arrows. Note that the signal peptide precedes domain 1. Sites of potential N-linked glycosylation are double underlined. Sites of potential dibasic endoproteolytic cleavage are in boldface.



The conceptual amino acid sequence consisted of 1251 amino acids with an estimated pI of 5.92, a predicted molecular mass of 134,710 Da, and five potential N-linked glycosylation sites. RGD and laminin B2 chain cell adhesion sequences were not identified. Northern blot analysis of mouse embryo RNA using a 3`-untranslated region probe identified a transcript band of 4.6 kilobases (data not shown). In this regard, 4310 nucleotides have been isolated by cDNA cloning, including a 3`-untranslated region of 401 nucleotides and a 5`-upstream sequence of 156 nucleotides. The apparent discrepancy between the Northern analysis result and the cDNA sequence analysis suggested that the 5`-upstream sequence may include 300 nucleotides of additional upstream sequence. This estimate was consistent with preliminary primer extension mapping studies indicating that the 5`-upstream sequence is 400-500 nucleotides in length.()

A total of 19 cysteine-rich repeats were found in domains 2 and 4 of the new mouse gene product. Thirteen were EGF-like and 11/13 showed the calcium binding consensus sequence: (D/N)(I/V)(D/N)(E/D)C. This consensus was derived from an analysis of 154 EGF-CB repeats in 23 different proteins and from structural analyses of the EGF-CB repeat, both bound and unbound to calcium ion (Selander-Sunnerhagen et al., 1992). Variations on the consensus have been noted previously (Yin et al., 1995), and one of these, DL(N/D)EC, was identified in the third EGF-like repeat of domain 4. In addition, a potential calcium binding sequence, which has not previously been reported (ET(N/D)EC), was identified in the first EGF-like repeat of domain 4. Ten of 13 EGF-CB repeats also contained a second consensus sequence, C X(D/N) XXXX(Y/F) XC, which represents a recognition sequence for an Asp/Asn hydroxylase that co- and posttranslationally modifies D/N residues (Stenflo et al., 1987; Gronke et al., 1989).

We (Pereira et al., 1993; Zhang et al., 1994; Yin et al., 1995) and others (Kanzaki et al., 1990; Taipale et al., 1994) have previously noted that similarities exist in the structural organization of LTBP and fibrillin. Fibrillin-1 and fibrillin-2 are secreted extracellular matrix proteins with five structurally distinct domains (cysteine-rich repeats are the dominant motif in two of five domains). Like the fibrillins, LTBP also can be divided into five structurally distinct domains. As shown in Fig. 2for LTBP-1, these include a relatively short domain downstream of the signal peptide with an estimated pI of 11.14 (amino acids 21-33; domain 1); a domain consisting of EGF-like, EGF-CB, TGF-bp, and Fib motifs plus a proline-rich and glycine-rich sequence (amino acids 34-407; domain 2); a proline-rich domain (amino acids 408-545; domain 3); a large, domain consisting of EGF-CB, TGF-bp, and TGF-bp-like repeat motifs (amino acids 546-1379; domain 4); and a relatively short domain at the carboxyl terminus (amino acids 1380-1394; domain 5). These similarities in fibrillin and LTBP likely explain the initial isolation of the low homology mouse PCR fragment ( i.e. the human fibrillin-1 oligonucleotide primers used to amplify mouse cDNA were designed to direct the synthesis of a EGF-CB repeat).

Evidence That the Mouse Gene Codes for a New Latent TGF- Binding Protein

An initial goal was to determine if the mouse gene we had cloned was a third member of the fibrillin gene family or a third LTBP. Comparison of sequence identity using the GAP and BESTFIT programs (Genetics Computer Group) revealed only 27% amino acid homology between the new mouse gene product and fibrillin-1. A similar level of sequence identity (22%) existed between the mouse gene product and human fibrillin-2. These values are low for a putative fibrillin family member, since fibrillin-1 and fibrillin-2 share 50% identity (Zhang et al., 1994). A search of available data bases revealed that the mouse gene product was most similar to human and rat LTBP-1 and, especially, to human LTBP-1. Amino acid sequence comparison with human LTBP-1 showed 60% identity for domain 1; 52% identity for domain 2; 30% identity for domain 3; 43% identity for domain 4; and 7% identity for domain 5. The average identity over the five domains was 38.4%. Significantly, cysteine residues in the mouse gene product and human LTBP-1 were highly conserved.

Recent evidence suggests that the fibrillin and LTBP genes can be distinguished on the basis of their expression patterns during development. Whereas the fibrillins are exclusively expressed by connective cells (Zhang et al., 1994),LTBP is co-expressed with TGF- in epithelial, parenchymal, and connective cells (Tsuji et al., 1990). If the sequence identity comparison was correct in suggesting that the new mouse gene was a LTBP family member, it followed that all three cell types would express this gene. Tissue in situ hybridization was used to test this hypothesis.

An overview of the expression pattern is presented in Fig. 3. Approximate midsagittal sections of normal mouse embryos at days 8.5-9.0, 13.5, and 16.5 p.c. of development were hybridized with a S-labeled single-stranded antisense riboprobe synthesized from a 382-base pair cDNA coding for the 3`-untranslated region of the mouse gene. The probe sequence showed less than 30% identity when compared with the corresponding sequence of human LTBP-1 and LTBP-2, which is too low to give spurious hybridization signals with the level of stringency we employed. Uterine and placental tissues were also present in the day 8.5-9.0 embryo tissue sections. As a negative control, a S-labeled single stranded normal sense riboprobe from the same cDNA construct was used. At day 8.5-9.0 of development, expression of the new mouse gene above the background of the experiment was observed in mouse embryo tissues; the mesometrial and anti-mesometrial uterine tissues; and the ectoplacental cone, placenta, placental membranes. Expression of the transcript above the background of the experiment was also observed in mouse embryo tissues at days 13.5 and 16.5 p.c. Gene expression was particularly intense in the liver.


Figure 3: Overview of expression of the LTBP-3 gene during development, as determined by tissue in situ hybridization. The figure consists of autoradiograms made by direct exposure of tissue sections to film after hybridization with radiolabeled probes but before dipping slides in radiographic emulsion. Day 8.5-9.0 sections contained embryos surrounded by intact membranes, uterine tissues, and the placental disk, cut in random planes. Day 13.5 and 16.5 p.c. sections contain isolated whole embryos sectioned in the sagittal plane near or about the mid-line. Arrowheads point to liver (day 13.5 p.c. embryo on the left) and to brain and spinal cord (day 13.5 p.c. embryo on the right and day 16.5 p.c. embryo). Identical conditions were maintained throughout autoradiography and photography, thereby allowing a comparison of the overall strength of hybridization in all tissue sections (days 8.5 versus 13.5 versus 16.5 p.c., and antisense versus sense negative control probes).



Microscopy of day 8.5-9.0 embryos p.c. (which was taken from the same slides used to prepare whole mount sections shown in Fig. 3) confirmed the widespread expression of the new mouse gene by mesenchymal cells (data not shown). Significant expression in the developing central nervous system, somites, and cardiovascular tissue (myocardium plus endocardium) was observed (Fig. 4, A and B). This pattern of expression contrasts sharply with that of fibrillin-1, which is expressed only in mouse endocardium at this stage of development.


Figure 4: Selected microscopic views of mouse LTBP-3 gene expression in day 8.5-9.0 p.c. mouse developing tissues. All photographs were taken from the same slides used to prepare whole mount sections shown in Fig. 3 (after dipping slides in radiographic emulsion). A, neural tube; note the expression by neuroepithelial cells and by surrounding mesenchyme. B, heart. The figure demonstrates expression by myocardial and endocardial ( arrowheads) cells. Darkfield photomicrographs were taken after exposure of tissues to photographic emulsion for 2 weeks. In all darkfield photographs, red blood cell and other plasma membranes give a faint white signal that contributes to the background of the experiment. 1 cm = 20 µm (all figures).



Microscopy of day 13.5 and day 16.5 p.c. embryos (also taken from the same slides used to prepare whole mount sections shown in Fig. 3) demonstrated expression of the new mouse gene by osteoblasts and by periosteal cells of the calvarium, mandible, and maxilla (Fig. 5 A). The transcript was also identified in both cartilage and bone of the lower extremity (data not shown). A positive signal was detected in perichondrial cells and chondrocytes of articular cartilage, the presumptive growth plate, the cartilage model within the central canal, blood vessel endothelial cells within the mid-diaphysis, and the surrounding skeletal muscle cells (not shown). The new mouse gene was also expressed by respiratory epithelial cells lining developing small airways and connective tissue cells in the pulmonary interstitium (Fig. 5 B, upper panel) and by myocardial cells (atria and ventricles) and endocardial cushion tissue (Fig. 5 B, lower panel). A positive signal was also expressed by cells within the walls of large arteries (data not shown). The transcript was also expressed by acinar cells of the exocrine pancreas (Fig. 5 C, upper panel). Mucosal epithelial cells lining the upper and lower digestive tract also expressed the transcript (Fig. 5 C, lower panel), as did the smooth muscle and connective tissue cells found in the gut submucosa and the parenchymal cells (hepatocytes) and extramedullary hematopoietic cells of the liver (not shown). The new mouse gene was also expressed by cells of developing nephrons, the ureteric bud, kidney blastema, and the kidney interstitium (Fig. 5 D, upper panel), by epidermal and adnexal keratinocytes, dermal connective tissue cells (Fig. 5 D, lower panel), and brown fat cells within the dorsal subcutis (data not shown) and by ganglion cells of the developing mouse retina (Fig. 6 E). The transcript was also intensely expressed by cells within the cerebrum, brainstem, spinal cord, and peripheral nerves (not shown). In summary, Figs. 3-5 clearly demonstrated expression of the new mouse gene by epithelial, parenchymal, and connective tissue cells.


Figure 5: Microscopy of mouse LTBP-3 gene expression in day 13.5 and day 16 p.c. mouse developing tissues. All photographs were taken from the same slides used to prepare whole mount sections shown in Fig. 3 (after dipping slides in radiographic emulsion). A, cartilage model of developing long bone from lower extremity. Note expression by chondrocytes and by perichondrial cells. B, lung and heart. In the upper panel (lung), note expression by epithelial cells of developing airway and by the surrounding parenchymal cells. The lower panel (heart) demonstrates continuing expression by myocardial cells. C, digestive system. In the upper panel (pancreas), note expression by acinar epithelial cells. The lower panel demonstrates expression by intestinal epithelial and subepithelial cells. D, kidney and skin. In the upper panel (kidney), note expression by blastemal cells beneath the kidney capsule, epithelial cells of developing nephrons and tubules, and the interstitial mesenchyme. The lower panel demonstrates expression by epidermal, adnexal, and dermal cells of developing skin. E, retina. Note expression by retinal epithelial cells and by adjacent connective tissue cells. Darkfield photomicrographs were taken after exposure of tissues to photographic emulsion for 2 weeks. In all darkfield photographs, red blood cell and other plasma membranes give a faint white signal that contributes to the background of the experiment. Note the absence of spurious hybridization signal in areas of the slide that lack cellular elements. 1 cm = 20 µm (all figures).




Figure 6: Time-dependent expression of the LTBP-3 gene by MC3T3-E1 cells. mRNA preparation and Northern blotting were performed as described under ``Experimental Procedures.'' Aliquots of total RNA as determined by UV spectroscopy were loaded in each lane of the Northern gel (not shown). As demonstrated by methylene blue staining (Sambrook et al., 1989), equal amounts of RNA were transferred to the nylon membrane (not shown). The results demonstrate a clear, strong peak in LTBP-3 gene expression by 14 days in culture. Weaker signals denoting LTBP-3 gene expression also can be observed after 5 days and 28 days in culture.



Collectively, the sequence identity and gene expression data favor the conclusion that the new mouse gene product was an LTBP family member. To further support this conclusion, MC3T3-E1 mouse preosteoblasts were used to demonstrate that the mouse gene product was capable of binding TGF-. MC3T3-E1 cells were chosen because previous studies have shown that they synthesize and secrete TGF-, which may act as an autocrine regulator of osteoblast proliferation (Amarnani et al., 1993; Van Vlasselaer et al., 1994; Lopez-Casillas et al., 1994).

We first asked whether or not MC3T3-E1 cells co-express the mouse gene product and TGF-. Cells were plated on 100-mm dishes under differentiating conditions (Franseochi et al. 1992; Quarles et al., 1992) and the medium was replaced twice weekly. Parallel dishes were plated and assayed for cell number and alkaline phosphatase activity, which confirmed that osteoblast differentiation was indeed taking place. Aliquots of total cellular RNA was prepared from these MC3T3-E1 cells after 5, 14, and 28 days in culture for Northern blot analysis. As shown in Fig. 6, expression of the new mouse gene peaked on day 14 of culture. This result has been independently reproduced (data not shown). Since MC3T3-E1 cells also show a peak in alkaline phosphatase activity on day 14 of culture (Quarles et al., 1992), the results suggest for the first time that LTBP-2 gene expression is an early marker of osteoblast differentiation.

We then prepared an affinity-purified antibody (274) capable of immunoprecipitating the new mouse gene product (see ``Experimental Procedures''). A full-length mouse cDNA was assembled into the pcDNA3 mammalian expression vector (Invitrogen) and expressed following transient transfection of 293T cells. Nascent polypeptides, radiolabeled by addition of [S]Cys to the medium of transfected cells, were immunoprecipitated using affinity-purified antibody 274. As shown in Fig. 7 , the new mouse polypeptide was estimated to be 180-190 kDa. To ensure the specificity of antibody 274 binding, we showed that preincubation of antibody with 10 µg of synthetic peptide blocks immunoprecipitation of the 180-190 kDa band.


Figure 7: Antisera 274 specifically binds LTBP-3 epitopes. Transfection of 293T cells with a full-length mouse LTBP-3 expression plasmid followed by radiolabeling, preparation of medium samples, immunoprecipitation, and 4-18% gradient SDS-polyacrylamide gel electrophoresis were performed as described under ``Experimental Procedures.'' The figure presents a SDS-polyacrylamide gel electrophoresis autoradiogram of medium samples following a 2-day exposure to film. Lane assignments are as follows: lane 1, radiolabeled 293T medium (prior to transfection) immunoprecipitated with preimmune serum; lane 2, radiolabeled 293T medium (prior to transfection) immunoprecipitated with antibody 274; lane 3, radiolabeled 393T medium (following transfection and preincubation with 10 µg of LTBP-3 synthetic peptide mixture) immunoprecipitated with antibody #274; lane 4, radiolabeled 293T medium (following transfection) immunoprecipitated with antibody 274. As indicated by the bar, the full-length LTBP-3 molecule migrated at 180-190 kDa.



Finally, MC3T3-E1 cells were cultured for 7 days under differentiating conditions and double-labeled with 30 µCi/ml [S]cysteine and [S]methionine in deficient media. Radiolabeled media was dialyzed into cold PBS with protease inhibitors. Aliquots of the dialyzed medium sample (10incorporated CPM) were analyzed by a combined immunoprecipitation/Western analysis protocol. The mouse polypeptide was clearly and reproducibly secreted by MC3T3-E1 cells, migrating under reducing conditions as a single band of 180-190 kDa (Fig. 8, top panel). Consistent with the results of previous studies ( e.g. Miyazono et al. (1988), Dallas et al. (1994), Moren et al. (1994)), bands of 70 and 50 kDa corresponding to the TGF-1 precursor were co-immunoprecipitated with the 180-kDa LTBP-3 protein. Weak bands of 40 and 12 kDa were also identified in experiments in which only immunoprecipitation was performed (data not shown). The latter were not included in the top panel of Fig. 8 because they migrated within that portion of the gel included in the Western analysis. Protein bands of 70-12.5 kDa are not variant forms of LTBP-3; Fig. 7demonstrates that LTBP-3 migrates as a single band of 180-190 kDa following transient transfection of 293T cells, which fail to make TGF-1 (data not shown). As shown in Fig. 8( bottom panel), we also found a unique band consistent with monomeric mature TGF-1 in the LTBP-2 immunoprecipitate. Antibody 274 is incapable of binding TGF-1 as determined by radioimmunoassay using commercially available reagents (R& Systems) and the manufacturer's suggested protocols.These results have been reproduced in six independent experiments that utilized three separate lots of MC3T3-E1 medium. Altogether, the results presented in Figs. 6 and 8 demonstrate that the new mouse LTBP-like polypeptide binds TGF- in vitro.


Figure 8: Co-immunoprecipitation of LTBP-3 and TGF-1 produced by MC3T3-E1 cells. Top panel, aliquots (10incorporated counts/min) of radiolabeled media produced by MC3T3-E1 cells after 7 days in culture were immunoprecipitated as described under ``Experimental Procedures.'' Bars indicate the position of cold molecular mass standards used to estimate molecular mass: 200, 97.4, 69, 46, 30, 21.5, and 14.3 kDa (Rainbow mix, Amersham Corp.). Immunoprecipitates were separated using 4-18% gradient SDS-polyacrylamide gel electrophoresis and reducing conditions. The figure shows a negative control ( left lane) consisting of MC3T3-E1 medium immunoprecipitated using preimmune sera. In contrast, the right lane consists of MC3T3-E1 medium immunoprecipitated with anti-LTBP-3 antibody #274. Bottom panel, Western blotting was performed as described (see ``Experimental Procedures'') using the lower portion of the gradient gel and a commercially available antibody to TGF-1 (Santa Cruz Biotechnology, Inc.). Antibody staining was detected using commercially available reagents and protocols (ECL Western blotting Reagent, Amersham Corp.). The figure shows the negative (preimmune sera) control ( left lane). The right lane consists of MC3T3-E1 medium immunoprecipitated with anti-LTBP-2 antibody 274. The intense band at the top of the photo ( right and left lanes) represents antibody light chains detected by the ECL reagent.




DISCUSSION

This paper reports the molecular cloning of a novel gene in the mouse that encodes a single transcript of 4.6 kilobases and a deduced polypeptide of 1251 amino acids. Although it is similar to fibrillin, the amino acid sequence of the new mouse gene product most resembles the latent TGF- binding protein. Additionally, the mouse gene is expressed more widely during development than either fibrillin-1 or fibrillin-2. The fibrillin-1 and fibrillin-2 genes are expressed by mesenchymal cells of developing connective tissue (Zhang et al., 1994),while the TGF- genes and proteins are expressed by developing epithelial, parenchymal, and stromal cells (Heine et al., 1987; Lehnert and Akhurst, 1988; Pelton et al., 1989, 1990a, 1990b; Millan et al., 1991). Tsuji et al. (1990) and others have suggested that the expression of TGF- binding proteins should mirror that of TGF- itself. As shown in Figs. 3-5, the mouse gene is intensely expressed by epithelial, parenchymal, and stromal cells during murine development, a pattern that is consistent with the co-expression hypothesis. Finally, the new mouse gene product also binds TGF- produced by MC3T3-E1 cells. Collectively, these data indicate that we have isolated a new member of the LTBP gene family, which we designate LTBP-3.

No previous study has presented a mouse LTBP sequence, but it seems clear that the gene reported in this paper is not simply the mouse homologue of human LTBP-1 or LTBP-2. First, domain 4 of the LTBP-3 coding sequence has a smaller number of EGF-like repeat motifs than reported for either LTBP-1 and LTBP-2 (LTBP-3 = 8; LTBP-2 = 13; and LTBP-1 = 11). Second, we have isolated several fragments of the human LTBP-3 gene and shown that the coding sequence is distinct from that of LTBP-1 and LTBP-2 (data not shown). Third, the human LTBP-1, LTBP-2, and LTBP-3 genes are localized to separate chromosomes. The LTBP-1 gene was assigned to human chromosome 2 based on the analysis of human x rodent somatic cell hybrid lines (Stenman et al., 1994); the LTBP-2 gene was assigned to human chromosome 14, band q24 based on the analysis of human x rodent somatic cell hybrid lines and fluorescent in situ hybridization studies (Moren et al., 1994); and the LTBP-3 gene was localized to chromosome 11, band q12 (while the mouse gene was mapped to mouse chromosome 19, band B, a region of conserved synteny) from the analysis of human x rodent somatic cell hybrid lines and fluorescent in situ hybridization studies (Li et al., 1995).

If LTBP-3 is like LTBP-1, it has the potential to function as a secreted, extracellular structural protein. As demonstrated here, domain 1 of LTBP-3 appears to be a unique sequence that likely has a globular conformation. Domain 1 also is highly basic and may facilitate LTBP-2 binding to acidic molecules ( e.g. acidic proteoglycans) within the extracellular space. Sequences rich in basic amino acids have also been shown to function as endoproteolytic processing signals for several peptide hormones (Barr, 1991; Steiner et al., 1992). It is possible, therefore, that the NHterminus of LTBP-3 is proteolytically processed in a tissue-specific manner. Domains 2 and 4 consist of consecutive cysteine-rich repeats, the majority of which are of the EGF-CB type. Besides binding calcium, these repeats may provide LTBP-3 with regions of extended conformation capable of interacting with other matrix macromolecules (Engel, 1989). Domain 3 is proline-rich and may be capable of bending (or functioning like a hinge) in three-dimensional space (MacArthur and Thornton, 1991). (In this regard, domain 2 is of interest because it has a similar stretch of 135 amino acids that is both proline- and glycine-rich. Since glycine-rich sequences are also thought to be capable of bending or functioning like a hinge in three-dimensional space, this amino acid sequence may interrupt the extended conformation of domain 2, thereby providing it with a certain degree of flexibility.) Domain 5 also appears to be a unique sequence having a globular conformation. The absence of a known cell attachment motif may indicate that, in contrast to LTBP-1, the LTBP-3 molecule may have a more limited role in the extracellular matrix ( i.e. that of a structural protein) in addition to its ability to target latent TGF- complexes to specific connective tissues.

Our data indicate that MC3T3-E1 preosteoblasts co-express LTBP-3 and TGF-1 and that these proteins form a complex in the culture medium. While the nature of the complex is currently under investigation, these results are particularly interesting because bone represents one of the largest known repositories of latent TGF- (200 µg/kg bone; Seyedin et al., 1986, 1987), and because this growth factor plays a critical role in the determination of bone structure and function. For example, TGF- is thought to (i) provide a powerful stimulus to bone formation in developing tissues, (ii) function as a possible ``coupling factor'' during bone remodeling (a process that coordinates bone resorption and formation), and (iii) exert a powerful bone inductive stimulus following fracture. Activation of the latent complex may be an important step governing TGF- effects, and LTBP may modulate the activation process ( e.g. it may ``protect'' small latent complexes from proteolytic attack); therefore, further characterization of LTBP in skeletal tissues should provide important new insights into bone structure and function.

The idea that LTBP may regulate TGF- activity by modulating the intracellular assembly and extracellular storage of latent growth factor complexes must remain speculative, however, because the function of LTBP in bone is uncertain at present. Bonewald et al. (1991) showed that the major form of TGF- in conditioned media from bone organ cultures is a 100-kDa (small) latent complex lacking LTBP. (Chinese hamster ovary cells also produce only the 100-kDa latent complex.) More recently, Dallas et al. (1994) demonstrated that cultured osteoblast-like cells derived from osteosarcomas of various types secrete two major forms of latent TGF-1, namely, a 290-kDa complex that contains 190-kDa LTBP-1 and a 100-kDa complex lacking LTBP-1. A second high molecular mass complex that contained latent TGF-2 and LTBP was also identified. Even though the presence and relative amount of low and high molecular mass complexes varied with cell type, the 100-kDa latent TGF- complex appeared to be the physiologically important form in bone cells because TGF-1 and LTBP were not coordinately expressed, and LTBP was not required for the assembly and secretion of small latent complexes.

We offer two explanations for the apparent difference between our results and those of Dallas et al. (1994). On one hand, the differences may reflect trivial species differences in reagents used by the two groups ( i.e. mouse versus rat/human). Alternatively, expression of large latent TGF- complexes bearing LTBP may be physiologically relevant to, i.e. may be part of the mechanism of, the preosteoblast osteoblast differentiation cascade. We base the second possibility on our evidence that MC3T3-E1 cells express large latent TGF-1 complexes bearing LTBP-2 precisely at the time of transition from the preosteoblast to osteoblast phenotype (day 14 in culture or at the onset of alkaline phosphatase expression; see Quarles et al. (1992)). Dallas et al. (1994) may not have found large latent complexes bearing LTBP because their cell and organ culture systems do not truly model osteoblast differentiation. The organ culture model, for example, likely is composed of differentiated osteoblasts but few bone progenitors, making it a difficult model at best in which to study the differentiation cascade. It is also well known that MG63, ROS17/2.8, and UMR 106 cells are rapidly dividing and they express the osteoblast phenotype. Thus, these osteoblast-like cell lines do not show the uncoupling of cell proliferation and cell differentiation that characterizes the normal (physiologically relevant) preosteoblast osteoblast transition (Gerstenfeld et al., 1987; Stein and Lian, 1993). Therefore, the production of small versus large latent TGF- complexes may be associated with specific stages in the maturation of bone cells.

It will be important for future studies to also determine if LTBP-3 binds calcium, since EGF-CB repeats have been shown to mediate high affinity calcium binding in LTBP-1 and other proteins (Colosetti et al., 1993). Calcium binding, in turn, may contribute to molecular conformation and the regulation of its interactions with other molecules. The presence of dibasic amino acids suggests that LTBP-3 may also undergo cell- and tissue-specific proteolysis. Finally, it will be important for future studies to identify LTBP isoforms and their functional role during tissue morphogenesis and wound healing. TGF- regulates extracellular matrix production by suppressing matrix degradation (through a decrease in the expression of proteases such as collagenase, plasminogen activator, and stromelysin plus an increase in the expression of proteinase inhibitors such as plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase-1) and by stimulating matrix macromolecule synthesis (for recent reviews, see Lyons and Moses (1990), and Miyazono et al. (1993)). Conversely, production of extracellular matrix has been shown to down regulate TGF- gene expression (Streuli et al., 1993). TGF- may therefore regulate extracellular matrix production through a sophisticated feedback loop that influences the expression of a relatively large number of genes. LTBP-1, LTBP-2, and LTBP-3 may contribute to this regulation by facilitating the assembly and secretion of large latent growth factor complexes and then targeting the complex to specific connective tissues (Taipale et al., 1994).


FOOTNOTES

*
This work was supported in part by the National Institutes of Health (HL-41926 and AR-40586) and by a grant from the Lucille B. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L40459.

§
To whom correspondence should be addressed: Dept. of Pathology, University of Michigan, MSRB I, Rm. 3520, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0650. Tel.: 313-747-4774; Fax: 313-936-9353; E-mail: Jeff_Bonadio@med.umich.edu.

The abbreviations used are: TGF-, transforming growth factor-; LTBP, latent TGF- binding protein; EGF, epidermal growth factor; EGF-CB, EGF-like repeats with the potential to bind calcium; TGF-bp, a cysteine-rich motif initially identified in the transforming growth factor-1-binding protein; Fib motif, a cysteine-rich motif initially identified in fibrillin-1; PCR, polymerase chain reaction; nt, nucleotide(s); PBS, phosphate-buffered saline; p.c, postcoital.

The paper by Li et al. (1995), which refers to the LTBP-2 gene, was in press at the time that the paper describing the human LTBP-2 gene by Moren et al. (1994) was published; these genes are the same.

W. Yin, E. Smiley, and J. Bonadio, unpublished results.


ACKNOWLEDGEMENTS

The authors are grateful to F. Ramirez for advice and discussion of results prior to publication.


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