THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biolow, Inc.

Vol. 267, No. 5, Issue of February 15, pp. 3274-3281, 1992 Printed in U.S.A.

Characterization of Mouse Thrombospondin 2 Sequence and Expression during Cell Growth and Development*

(Received for publication, June 28,1991)

Carol D. LahertySB, Karen O’RourkeS, Frederick W. Wolf$, Ronald Katz$$, Michael F. Seldinll, and Vishva M. Dixit$§ 11 From the $Department of Pathology and the §Cellular and Molecular Biology Program, The University of Michigan Medical School. A n n Arbor. Michigan 48109 and the TDeDartments of Medicine and Microbiology, Duke University Medical Center, Durham, North Carolina 127710

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose expression has been associated with a variety of cellular processes including growth and embryogenesis. The recent discovery of the exist- ence of a second mouse TSP gene necessitates careful examination of the discrete biochemical and functional properties associated with each molecule. In this re- port, the primary structures of human TSP, mouse TSPl (mTSPl), mouse TSP2 (mTSP2), and chicken TSP are compared; and the expression of mTSPl and mTSP2 during embryogenesis and growth factor-me- diated cell proliferation is examined. The cloning and sequencing of the entire coding regions of mTSPl and mTSP2 revealed considerable conservation of residues critical for TSP structure and function; these data suggest that TSPS is capable of trimer formation and many of the same cell-surface and ligand interactions that mediate TSP function. Comparison of the various TSP sequences also allowed the assignment based on sequence homology of previously reported human TSP as TSPl and chicken TSP as TSP2. mTSP2, like mTSP1, was shown to be a primary response gene when quiescent Swiss 3T3 cells were stimulated with serum, platelet-derived growth factor BB, basic fibro- blast growth factor, or interleukin-la. Interestingly, TSPl and TSPS exhibited markedly different tissue- and stage-specific patterns of mRNA expression dur- ing mouse embryogenesis, implying that the two TSP molecules possess discrete functional properties impor- tant for development. Additionally, the TSP genes (Thbsl and Thbs2) were mapped to single loci on mouse chromosomes 2 and 17, respectively.

Thrombospondin (TSP)’ is a trimeric glycoprotein with an M, of 450,000 that is synthesized, secreted, and transiently

HL-39037 (to V. M. D.), HG-00101 (to M. F. S.), and DE-00301-01 * This work was supported by National Institutes of Health Grants

(to R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I( Established Investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed Dept. of Pathology, The University of Michigan Medical School, 1301 Catherine St., Box 0602, Ann Arbor, MI 48109-0602. Tel.: 313-747- 2921; Fax: 313-764-4308.

The abbreviations used are: TSP, thrombospondin (h, m, and ch prefixes represent human, mouse, and chicken, respectively); PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; Il-I@, interleukin-I@; cM, centimorgans; SDS, sodium dodecyl sulfate; kb, kilobase pair(s); bp, base pair(s).

incorporated into the extracellular matrix by a variety of cells including fibroblasts, smooth muscle cells, endothelial cells, and keratinocytes (Refs. 1 and 2; reviewed in Ref. 3). Each TSP subunit is composed of multiple discrete domains that mediate TSP binding to a variety of ligands including heparin, collagen, fibronectin, plasminogen, and plasminogen activator (reviewed in Refs. 4 and 5). Previous work has also demon- strated the presence of two distinct TSP receptors (6). Pre- sumably, it is through domain-specific interactions with these ligands and cell-surface receptors that TSP influences mul- tiiple cellular processes such as cell attachment, migration, and proliferation.

Evidence clearly suggests that TSP plays an important role in the growth response of mesenchymal cells. Cell surface- associated TSP facilitates the mitogenic response of smooth muscle cells to epidermal growth factor (7). In contrast, agents such as heparin (8) and anti-TSP monoclonal antibodies (9) that block TSP’s interactions with other matrix molecules and the cell surface are strong inhibitors of smooth muscle cell growth. The biosynthesis of TSP by smooth muscle cells and fibroblasts is regulated by the mitogens platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF), respectively (10, 11). When quiescent cells are treated with PDGF or FGF, TSP mRNA levels are rapidly induced without concomitant new protein synthesis (11, 12). In fact, simultaneous treatment with growth factor and the protein synthesis inhibitor cycloheximide causes superinduction of TSP mRNA (12). Accumulating evidence suggests that TSP induction is directly related to mitogenesis. PDGF induction of TSP in smooth muscle cells parallels the mitogenic re- sponse (12), and FGF induction of TSP mRNA in mouse fibroblasts occurs only in cell lines for which FGF is mitogenic (11). Therefore, TSP mRNA levels are regulated by PDGF and FGF in a manner similar to c-fos and other “immediate- early” growth regulatory genes (13), and TSP may be consid- ered a member of the competence gene family that mediates the mitogenic response of cells to PDGF and other growth factors (14, 15).

TSP synthesis is associated with all stages of embryogenesis and development (16, 17). TSP exhibits tissue- and stage- specific patterns of expression in the developing mouse em- bryo (17). TSP expression is generally highest in tissues in which cells are actively undergoing proliferation and migra- tion (17). In contrast, TSP expression declines following tissue differentiation, and TSP expression is much more restricted in adult tissue (17, 18).

Until recently, the many and sometimes conflicting prop- erties and functions associated with TSP were attributed to a single TSP molecule. However, the recent discovery of a second expressed TSP gene (19) necessitates careful re-ex-

3274

Mouse Thrombospondin 2 Sequence and Expression 3275

amination of TSP physical properties, expression, and asso- ciated functions as it is likely that the different cellular processes associated with TSP are mediated by different TSP molecules or by differences in the relative amount of each TSP molecule. In this study, differences in the structure and expression of TSPl and TSP2 were examined. The sequences of the entire coding regions of mTSPl and mTSP2 were determined, allowing a more complete comparison of the primary structures of hTSP, mTSP1, mTSP2, and chTSP. cDNA probes specific for mTSPl and mTSP2 were used to examine the discrete patterns of mTSPl and mTSP2 expres- sion during cell proliferation and mouse embryogenesis. mTSP2, like mTSP1, was demonstrated to be an immediate- early response gene in mitogen-stimulated Swiss 3T3 fibro- blasts, although subtle differences in the induction patterns of mTSP2 and mTSPl were observed. In the developing mouse embryo, mTSPl and mTSP2 exhibited strikingly dif- ferent tissue- and stage-specific patterns of expression, indi- cating the possibility of distinct and independent functions in embryogenesis, Finally, the genes for mTSPl and mTSP2 were mapped and found to reside on different mouse chro- mosomes.

MATERIALS AND METHODS

Cloning and Sequencing-A random-primed mouse day 17 embry- onic kidney cDNA library in the plasmid vector pcDNA (Invitrogen, San Diego, CA) was screened using 32P-labeled cDNA probes specific for mTSPl and mTSP2 as described previously (19). Plasmid from hybridizing colonies was prepared for sequence analysis by standard procedures including banding two times using a CsCl gradient. Plas- mid DNA was sequenced on both strands by the dideoxynucleotide method using modified T7 DNA polymerase (Sequenase, United States Biochemical Corp.) and synthetic oligonucleotides as primers (20). Sequence data were compiled and analyzed using Genetic Com- puter Group sequence analysis software version 6.2 (21).

Cell Culture-Swiss 3T3 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% calf bovine serum (HyClone Laboratories, Inc., Logan, UT). Cells were made quiescent by cultur- ing for 5-6 days in a 1:l mixture of Dulbecco’s Modified Eagle’s medium and Ham’s F-12 medium supplemented with 30 nM NazSe03 (ICN, Plainview, NY), 10 pg/ml human transferrin (Sigma), 0.2% bovine serum albumin (Sigma), and 10% reduced and alkylated fetal calf serum prepared as previously described (22). Quiescent cells were then stimulated with final concentrations of 20% calf bovine serum, or 20 ng/ml recombinant human IL-10 (gift of The Upjohn Co.) or 50 ng/ml recombinant human PDGF BB (gift of Amgen, Inc., Thou- sand Oaks, CA), or 25 ng/ml recombinant human basic FGF (gift of Synergen, Boulder, CO). When used, cycloheximide (Sigma) was added to cells at 10 pg/ml 30 min before serum or growth factors. Control treatment with cycloheximide alone also persisted 30 min longer than serum treatment.

DNA Probes-cDNA fragments used to probe the day 17 embryonic kidney library and Southern and Northern blots were gel-purified and 32P-labeled by the random-primed oligolabeling method (23). The mTSP1-specific probe spanned nucleotides 489-838, and the mTSP2- specific probe spanned nucleotides 470-826 of the previously de- scribed cDNA sequences (19). The nucleotide sequences defined by these probes represent the most divergent region of the two molecules, and no cross-hybridization was detected under the described experi- mental conditions (19). Other probes used included a 1.0-kb v-fos cDNA fragment (provided by E. Prochownik, The University of Michigan), a 2.0-kb Hind111 cDNA fragment of c-myc (also provided by E. Prochownik), and a 1.2-kb cDNA fragment of mouse thymidine kinase (provided by A. Pardee, Dana-Farber Cancer Institute).

RNA Isolation and Northern Blot Analysis-Total RNA was iso- lated from cultured cells using a previously described modification of the guanidinium method (24, 25). RNA (10 pg) was fractionated on 1% agarose gels containing 2.2 M formaldehyde, transferred to nitro- cellulose (Schleicher & Schuell), and hybridized with 5 X IO5 cpm/ml 32P-labeled cDNA fragments, Hybridizations were performed in 50% formamide, 5 X Denhardt’s solution (50 X is 1% Ficoll, 1% polyvi- nylpyrrolidone, and 1% bovine serum albumin), 0.1% SDS, 100 pg/ ml salmon sperm DNA, 5 X SSPE (1 X is 0.18 M NaC1,lO mM NaPO,

(pH 7.7), and 1 mM EDTA) at 42 “C for 12-24 h. Filters were washed twice in 2 X SSC (1 X is 0.15 M NaC1, 0.015 M sodium citrate (pH 7.0), 0.1% SDS for 10 min at room temperature; once in 0.1 X SSC, 0.1% SDS for 10 min at room temperature; and twice in 0.1 X SSC, 0.1% SDS for 30 min at 65 ‘C and subjected to autoradiography. The ethidium bromide staining of ribosomal RNA bands was examined both before and after transfer to filters to ensure equivalency of all lanes.

Mouse Developmental Northern Blots-Mus musculus strain CD-1 males were bred with virgin females. Embryonic day 1 was defined as the day of the appearance of a vaginal plug. Embryos were removed at the indicated time points, and individual organs and tissues were excised and snap-frozen in liquid nitrogen. Organs from littermates were pooled and homogenized in 6 M guanidinium chloride (Boehrin- ger Mannheim). Total RNA was isolated and subjected to electropho- resis as described. RNA was transferred to nylon membranes (Nytran, Schleicher & Schuell) and hybridized overnight with 5 x 10’ cpm/ml 32P-labeled DNA probes in 50% formamide, 1 X PE (1 X is 50 mM Tris (pH 7.5), 0.1% sodium pyrophosphate, 1% SDS, 0.2% polyvinyl- pyrrolidone, 2.2% Ficoll, 5 mM EDTA, and 1% bovine serum albu- min), 5 X SSC, and 150 pg/ml salmon sperm DNA. Blots were washed twice in 2 X SSC, 1% SDS for 10 min at room temperature and twice in 2 X SSC, 1% SDS for 30 min at 65 “C and subjected to autoradi- ography.

Chromosomal Localization-C3H/HeJ-gld/gM and Mus spretus (Spain) mice and (CBH/HeJ-gld/gld X M. spretus)F, X C3H/HeJ- gld/gM interspecific backcross mice were bred and maintained as previously described (26). DNA isolated from mouse organs by stand- ard techniques was digested with restriction endonucleases, and 10- pg samples were electrophoresed on 0.9% agarose gels. DNA was transferred to Nytran membranes, hybridized at 65 “C, and washed under stringent conditions as previously described (27). Probes in- cluded the mTSP1- and mTSP2-specific cDNA fragments and the previously described clones pshglu (28), PI11 1301 (29), pEo (30), and pTcrlO (31). Gene linkage was determined by segregation analysis. The most likely gene order was determined by analyzing all haplo- types and minimizing crossover frequency between all genes that were determined to be within a linkage group (32).

RESULTS

Isolation of mTSPl and mTSP2 cDNA Clones-Both mTSPl and mTSP2 cDNA clones were isolated by screening a mouse day 17 embryonic kidney cDNA library with mTSP1- and mTSP2-specific probes. Restriction mapping and South- ern blot analysis confirmed the identity of the clones as mTSPl or mTSP2; and several clones, including a 5.5-kb mTSPl clone and a 3.7-kb mTSP2 clone, were subsequently sequenced. The sequences have been submitted to GenBank’”. Sequence from the 5.5-kb mTSPl cDNA clone extended the predicted mTSP1 amino acid sequence from the originally reported 548 amino-terminal amino acids to 1170 amino acids encoding the entire mTSPl molecule. The 3.7-kb mouse TSP2 cDNA clone contained the entire coding region and an addi- tional 218 bp of 5”untranslated sequence and 370 bp of 3‘- untranslated sequence, thus extending the predicted mTSP2 amino acid sequence from the previously reported 873 amino- terminal amino acids to include the entire molecule of 1172 amino acids.

Comparison of hTSP, mTSP1, mTSP2, and chTSP Se- quences-Fig. 1 compares the derived amino acid sequences of hTSP, mTSP1, mTSP2, and the recently published chTSP (33). At first approximation, it was evident that there exists a gradient of homology among the TSP sequences. The amino terminus exhibited much less similarity than the carboxyl terminus; nevertheless, throughout all the TSP molecules, there was a striking conservation of cysteine residues. The sequence of the amino-terminal heparin-binding domain of hTSP is more similar to that of mTSPl than that of mTSP2 or chTSP. Over the first 250 amino acids, hTSP is 86% identical to mTSP1, but only 36% identical to mTSP2 and 38% similar to chTSP, suggesting that the reported hTSP sequence is the homolog of mTSP1. hTSP is thus referred to

3276 Mouse Thrombospondin 2 Sequence and Expression hTSPl M G mTSPl M F mTSP2 chTSP2 M L O R S R

96 96 92 98

hTSPl

mTSPl mTSPZ chTSP2

387 387 389 395

hTSPl mTSPI mTSP2 chTSP2

hTSPl mTSPl mTSP2 chTSP2

-

T A N M A N R T E K T E

Type II Repeal

IhTSPl mTSP1 mTSP2 chTSPZ

787 787 789 795

hTSPl mTSPl mTSP2 chTSP2

887 887 889 895

hTSPl mTSPl mTSP2 chTSP2

987 987 989 995

IhTSPl mTSPl mTSP2 chTSP2

1087 1087 1089 1095

hTSPl P mTSPl S mTSP2 A chTSP2 .I

FIG. 1. Comparison of derived amino acid sequences of hTSP1, mTSP1, mTSP2, and chTSP2. Periods signify gaps introduced to maximize similarity. Amino acid residues identical in two sequences are lightly shaded, and amino acid residues conserved in three or four sequences are darkly shaded. Conserved cysteines are boxed; other amino acid residues and functional motifs discussed under “Results” are boldly boxed. The signal sequence of hTSPl and those predicted for mTSP1, mTSP2, and chTSP2 are italicized. The 2 cysteines that participate in trimer formation are denoted by diamonds, and the cysteine that serves as a free sulfhydryl is denoted by an inverted triangle. Sequence domains are bracketed beneath the alignment, with dots denoting repeat element boundaries. Amino acid numbering is indicated to the right.

as hTSPl in this report. A putative human homolog of mTSP2 has been cloned,’ but its sequence is not yet available for comparison. In contrast, chTSP is more related to mTSP2 than to mTSP1, suggesting that it is the homolog of mTSP2. Over the first 250 amino acids, the chTSP sequence is 63% identical to mTSP2, but only 37% identical to mTSP1. chTSP is therefore referred to as chTSP2 throughout the remainder of this report. The sequence identities among the various TSPs over the first 250 amino acids are summarized in Table I.

Functional Domains Are Conserved among Various TSPs- Several functional domains through which TSP interacts with ligands and the cell surface and the amino acid residues

*T. Labell, P. McGookey-Milewicz, and P. Byers, manuscript in preparation.

1170 1170 1172 1778

TABLE I Percent identity among the various TSPs in the 250 amino-terminal

amino acids hTSPl mTSP1 mTSP2 chTSP2

hTSPl 100 86 36 38 mTSPl 100 33 37 mTSP2 100 63 chTSP 100

critical for maintaining TSP structure have been previously identified (4, 5 ) . Examination of the derived amino acid sequences of hTSP1, mTSP1, mTSP2, and chTSP2 revealed remarkable conservation of these regions between both dif- ferent species and the two different forms of TSP.

The amino-terminal sequence of mature human platelet

Mouse Thrombospondin 2 Sequence and Expression 3277

TSP, previously determined by protein sequencing (34), was identical to the presumed amino terminus of mTSPl over the first 10 amino acids. The preceding signal peptides were also substantially conserved (67% identity). The predicted amino- terminal sequences of the TSP2 molecules had hydrophobic character, suggesting that they code for signal peptide se- quences. Since the amino-terminal sequences of mature mTSP2 and chTSP2 are unknown, the predictive algorithm of von Heijne (35) was used to determine the most likely site of signal sequence cleavage. The amino-terminal sequence of mature mTSP2 was predicted to start with the glycine at position 19, which corresponds to the glutamic acid at position 23 of hTSP1; and mature chTSP2 was predicted to begin with the glutamine at position 23, corresponding to the isoleucine at position 21 of hTSP.

The amino terminus of TSP encodes the heparin-binding domain (34), which interacts with cell-surface sulfated gly- colipids (36, 37) and proteoglycans including syndecan (38, 39), and is capable of mediating attachment of cells to TSP. Two clusters of cationic amino acid residues in the heparin- binding domain implicated in the binding of anionic heparin (40) are indicated by the boxes located at positions 41-50 and 95-101 of hTSPl (Fig. 1). The first cluster was highly con- served between hTSPl and mTSP1. In contrast, in both mTSP2 and chTSP2, the arginine at position 46 was replaced with alanine, thereby reducing the cationic character of this stretch of residues. The second stretch of positively charged residues was also highly conserved between hTSPl and mTSPl as well as mTSP2. However, chTSP2 had a glutamic acid in place of lysine at position 99, thus changing the ionic character of this domain. Just distal to the heparin-binding domain are 2 cysteines at positions 288 and 292 of hTSPl that are involved in interchain disulfide linkage and trimer assembly (4)3; these cysteines were conserved among all the TSP molecules. Adjacent to these residues is the cysteine- rich procollagen homology domain, which is also found in the al-chain of type 1 procollagen and von Willebrand factor and has been postulated to facilitate TSP oligomerization (42). This domain, including all 10 cysteines, appeared to be con- served among the various TSPs, suggesting the existence of a common conformation.

Carboxyl-terminal of the procollagen homology domain are three TSP type 1 repeats that are conserved among all the TSPs and have also been identified in other proteins including complement components, thrombospondin-related anony- mous protein of Plasmodium falciparum, and circumsporozite protein of Plasmodium vivax (43). The peptide sequence VTCG, present in type 1 repeats, has been shown to mediate cell adhesion on circumsporozite protein (44). hTSPl and mTSPl contained two VTCG sequences, whereas mTSP2 and chTSP2 contained three such sequences within the type 1 repeats. The only divergence from the canonical VTCG sequence occurred in mTSP2, in which the last residue of the third repeat is an alanine instead of a glycine.

The TSP type 2 repeats contain 6 invariant cysteine resi- dues that are conserved among all TSPs examined. The spacing of these cysteines is similar to that found in mouse epidermal growth factor precursor (40), but the significance of this homology remains uncertain. However, the absolute conservation of these cysteines in all forms of TSP suggests they may be functionally important. The eight adjacent TSP type 3 repeats (five complete and three partial repeats) (40) are enriched in aspartic acid residues and constitute the calcium-binding domain of TSP (40). The conserved aspartic acid residues contribute the necessary oxygen atoms for cal-

K. O’Rourke and V. M. Dixit, unpublished data.

cium binding and thereby contribute to the calcium-depend- ent conformational change in TSP (45). Besides the critical aspartic acid residues, other amino acids including cysteines are highly conserved in the type 3 repeats, suggesting a similar conformation of this domain in all TSPs. The last type 3 repeat in both hTSPl and mTSPl contains an RGDA se- quence that, for hTSP1, has been shown to be recognized by integrin receptor avp3 and to mediate the attachment of endothelial cells to TSP (46). In mTSP2 and chTSP2, the alanine is conservatively substituted by an isoleucine so that this sequence becomes RGDI.

Carboxyl-terminal of the type 3 repeats lies a cysteine residue that acts as a free reactive sulfhydryl at position 1005 in hTSPl (47-49). This free sulfhydryl, which is conserved in mTSP1, is capable of forming disulfide-linked TSP oligomers (49) and complexes with other proteins including thrombin (50) and protease nexin 1 (51) and may represent a general mechanism for covalent incorporation into the cell surface/ matrix compartments. Surprisingly, this free sulfhydryl is abent in both mTSP2 and chTSP2, suggesting that these molecules may be incapable of forming intermolecular thiol- disulfide bonds between themselves or with other proteins. All the other cysteine residues in mTSP2 and chTSP2 align with those present in hTSPl and mTSPl and are presumably primarily involved in intrachain or interchain disulfide link- age.

Although there are a number of potential sites for N-linked glycosylation in hTSP1, only the asparagine residue at posi- tion 256 has been shown to be glycosylated (52). Whereas this glycosylation site is conserved in mTSP1, it is absent in both mTSP2 and chTSP2, suggesting differential post-transla- tional modification of TSPl and TSP2.

mTSP2 mRNA k v e k Increase in Response to Serum- Previous studies have demonstrated that hTSPl is a primary response gene when quiescent cells are stimulated with serum or a variety of growth factors (12). To determine if mTSP2 is also a primary serum response gene, quiescent Swiss 3T3 cells were treated with 20% calf bovine serum, and mTSPl and mTSP2 mRNA levels were examined by Northern blot analy- sis using cDNA probes specific for each. Results representa- tive of three individual experiments are shown in Fig. 2. mTSPl and mTSP2 mRNA levels were rapidly and tran- siently elevated in response to serum treatment. Accumula- tion of both mTSPl and mTSP2 mRNAs was maximal by 30 min of serum treatment, the earliest time point examined. However, mTSP2 mRNA levels declined to base line within 4-8 h, whereas elevated mTSPl mRNA levels persisted through 24 h of serum treatment. The levels of mTSPl and mTSP2 mRNAs present in quiescent cells and the rate at which mTSPl and mTSP2 mRNA levels declined following serum treatment varied somewhat between experiments, but the results shown remain representative. Variability in TSP mRNA levels in quiescent and cycling cells has been observed by several groups (11, 53) and may be due to differences in cell density or passage number. Treatment of quiescent cells with serum in the presence of cycloheximide resulted in mTSPl and mTSP2 mRNA inductions equal to or greater in magnitude than those observed with serum treatment alone. This is consistent with them both being primary response genes since new protein synthesis is not required for the induction of their transcripts. Cycloheximide superinduction of TSPl mRNA has been previously observed during serum and growth factor treatment of rat aortic smooth muscle cells and basic FGF and IL-1p treatment of Swiss 3T3 cells (11, 12, 54).

c-fos and c-myc mRNA levels were examined as controls

3278 Mouse Thrombospondin 2 Sequence and Expression

iTSP2

40s

:-myc

.-' TK

26s

FIG. 2. Northern blot analysis of to ta l RNA f rom serum- stimulated Swiss 3T3 cells. Quiescent Swiss 3T3 cells were treated with 20% calf bovine serum for the times indicated; dividing cells maintained in 10% calf bovine serum are labeled cycling cells. Si- multaneous or independent treatment with cycloheximide ( C H X ) for 2.5 h is also indicated. Northern blot analysis was performed as described under "Materials and Methods." Panels show the results of sequential probing with mTSP1, mTSP2, c-{os, c-myc, and thymidine kinase ( T K ) cDNA fragments. Autoradiographs were exposed for 48 h, except for c-myc and thymidine kinase, which were exposed for 5 days. EtBr staining of 28 S ribosomal RNA bands demonstrates equivalency of RNA loading.

for serum and cycloheximide induction since both genes dem- onstrate superinduction and are induced during the Go/Gl transition in the cell cycle (55,56). As expected, c-myc and c- fos mRNA levels were rapidly and transiently induced by serum and superinduced in the presence of cycloheximide, indicating that the cells were initially quiescent and then stimulated to traverse the Go/GI boundary and to enter the cell cycle. The kinetics of mTSP1, mTSP2, c-fos, and c-myc inductions were noticeably similar. mTSP1 and mTSP2, like c-fos, were rapidly induced, although the decline of mTSPl and mTSP2 mRNA levels more closely paralleled the decline of c-myc mRNA levels.

mTSPl and mTSP2 transcripts were present in an -2:l ratio in cycling Swiss 3T3 cells. However, the relative levels of mTSPl and mTSP2 mRNAs varied significantly among several other mouse fibroblast cell lines: The detection of thymidine kinase mRNA in subconfluent proliferating cells verified that a substantial number of the cells were cycling since thymidine kinase is only expressed during the late GI/ S boundary of the cell cycle (57). Similarly, the lack of detectable thymidine kinase mRNA in growth factor-deprived cells provided evidence that quiescence had been achieved.

mTSP2 mRNA Levels Increase in Response to PDGF BB, Basic FGF, and 11-IP-The response of mTSP1 and mTSP2 mRNA levels to PDGF BB, basic FGF, and IL-lP was ex- amined as it has been reported that TSPl mRNA levels increase in response to these factors (11, 12, 54). Northern blot analysis using probes specific for mTSPl or mTSP2 were performed using RNA from quiescent Swiss 3T3 cells stimu- lated with serum, PDGF BB, basic FGF, or IL-1P. As shown in Fig. 3, each growth factor caused increases in mTSPl and mTSP2 RNA levels equal to those observed during serum treatment. The magnitude and time course of growth factor inductions closely approximated those observed during serum induction (data not shown). Treatment with growth factors in the presence of cycloheximide also resulted in mTSP1 and mTSP2 mRNA inductions equal to or greater than those observed during treatment with growth factor alone.

' C. D. Laherty and V. M. Dixit, unpublished data.

serum PDGF FGF IL-1

- + - + - + - + ""

@ . ( b -a mTSPl

&.a @a *I) mTSP2

mmLp;;u FIG. 3. Northern blot analysis of

factor-stimulated Swiss 3T3 cells. Quiescent Swiss 3T3 cells were treated with 20% calf bovine serum, or 50 ng/ml PDGF BB, or 25 ng/ml basic FGF, or 20 ng/ml IL-lP for 30 min to 2 h; the time points of maximal induction of mTSPl and mTSP2 mRNAs are shown. Autoradiographs were exposed for 48 h. EtBr staining of 28 S ribo- somal RNA bands demonstrates equivalency of RNA loading.

placenta head body brain kidney 11 13 17 11 13 11 13 15 17 19 Ad 15 17 19 "

*.. " ' ;iur f l mTSP1

i. J d

b. b.. . d@w ' 4 1. mTSP2

r"""=, 4 h 4 2 8 s

hb . *" mTSP1

lung ltver heart 15 17 19 Ad 15 17 19 Ad 15 17 19 I d 2d Ad

*I ..*.a mTSP2

rn 2 8 s FIG. 4. Northern blot analysis of total RNA from mouse

embryonic tissues. The tissues shown were isolated from embryos and mice of the ages shown; numbers indicate embryonic age; I d = 1-day-old mice, 2d = 2-day-old mice, and Ad = adult mice. Total RNA was isolated, and Northern blot analysis was performed as described under "Materials and Methods." Autoradiographs were exposed to film for 2 weeks. EtBr staining of ribosomal RNA bands demonstrates the equivalency of loading; day 17 and adult liver lanes were underloaded by -50% as determined by scanning densitometry.

mTSPl and mTSP2 Are Differentially Expressed during Development-The tissue- and stage-specific expression of mTSPl and mTSP2 mRNAs during development was deter- mined by Northern blot analysis of RNA extracted from various tissues of the mouse embryo. mTSP1- and mTSP2- specific probes detected different patterns of expression of the two genes (Fig. 4). In the placenta, high levels of mTSP2 mRNA were detected from days 11 to 17 post-gestation. In contrast, mTSPSl was not detected until day 13 and was present at much lower levels than mTSP2. mTSPl and mTSP2 transcripts were detected in both the head and body a t day 13. However, mTSPl mRNA was evident by day 11, whereas mTSP2 mRNA was not detectable until day 13. High levels of both mTSPl and mTSP2 mRNAs were present in the lung at the earliest developmental stage examined (day 15) and persisted through adulthood. Similarly, both tran- scripts were constitutively expressed in the kidney from days 15 to 19. mTSPl and mTSP2 mRNAs were detected a t relatively low levels in the brain from day 15 to adulthood, with mTSP2 mRNA levels transiently increasing from days 17 to 19. The most striking example of differential tissue expression occurred in the liver. mTSPl mRNA was promi- nent at day 15, after which it decreased to barely detectable levels, whereas mTSP2 mRNA was only barely detectable from day 15 to adulthood. For this analysis, mTSPl and mTSP2 signals were standardized to 28 S EtBr staining to control for unequal RNA loading. In the heart, mTSPl mRNA

Mouse Thrombospondin 2 Sequence and Expression 3279

levels first detectable a t day 17 increased substantially follow- ing birth and declined in adults. mTSP2 message also in- creased substantially following birth, but the message re- mained a t high levels through adulthood.

Mouse Chromosomal Localization and Fine Mapping-To determine the chromosomal localization of the Thbs2 gene, a panel of DNA samples from an interspecific cross that had been characterized for >350 genetic markers throughout the mouse genome was utilized. The genetic markers included in this map span between 50 and 80 centimorgans on each mouse autosome and the X chromosome (for examples, see Refs. 58- 60). DNAs from the two parental mice (CSH/HeJ-gld/gld and (CBH/HeJ-gld/gld x M. spretw)F,) were digested with var- ious restriction endonucleases and hybridized with an mTSP2 probe to determine a restriction fragment length variation to allow haplotype analyses. An informative restriction fragment length variation was detected with PuuII-restricted DNAs (Fig. 5). Each of 114 PuuII-restricted DNAs from the (C3H/ HeJ-gld/gld x (C3H/HeJ-gld/gld x Mus spretus) Fl) interspe- cific backcross mice displayed either the homozygous (CC) or heterozygous (SC) F1 pattern when hybridized with the mTSP2 probe. Similarly, Thbsl was mapped using restriction fragment length variation analysis of PstI-restricted DNAs (Fig. 5).

The Thbs2 and Thbsl genes did not cosegregate in >40% of the backcross mice. Comparison of the haplotype distri- bution of the Thbs2 restriction fragment length variation indicated that in 113 of the 114 meiotic events examined, the Thbs2 locus co-segregated with t-complex protein 10b locus (Tcp-lob) (Fig. 6), a locus previously mapped to proximal mouse chromosome 17 (30). This result indicated that Thbs2 was linked with Tcp-lob (the probability of linkage is >0.99; upper 95% confidence limit by binomial distribution = 4.6 centimorgans (cM)). The haplotype distribution among other genes localized to mouse chromosome 17 is shown in Fig. 6. The best gene order (31) f S.D. (61) indicated that Thbs2 was located 0.9 f 0.9 cM distal to Tcp-lob and 7.0 & 2.4 cM proximal of the major histocompatibility locus H2eb. Simi- larly, the haplotype distribution of the Thbsl loci previously mapped to mouse chromosome 2, glucagon (Gcg), and inter-

Thbsl

6.6.

4.3.

-b

2.3. 2.1 .

Pstl

Thbs2

c

Pvull FIG. 5. Southern blot identification of Thbsl and Thbs2

restriction fragment length variations. The informative restric- tion digest is shown with the molecular size standards (in kilobases) indicated to the left. The arrows signify bands present in DNA from (CSH/HeJ-gld/gld X M. spretus)F1 (SC) mice that are not present in DNA from C3H/HeJ-gld/gH mice (CC) when subjected to PstI or PuuII restriction endonuclease digestion.

A Tcp-lob 0 I 0 0 Thbs2 1 0 0 0 H - E b I 0 0 I

39 66 0 1 3 5 Meiotic Events

1 C3H/HeJ-gld/g/d

(C3H/HeJ-g/d/g/dx Mus spretus) F1

B Gcg m n I o 1 0

Tbbsl I 0 0 I ma I 0 0 I 0 I

34 62 7 8 1 2 Meiotic Events

C3H/HeJ-g/d/g/d

0 (C3H/HeJ-g/d/g/dx Mus spretus) F1

FIG. 6. Segregation of Thbs2 among mouse chromosome 17 loci ( A ) and of Thbsl among mouse chromosome 2 loci ( B ) in (C3H/HeJ-gld/gld X M. spretus)F, X C3H/HeJ-gld/gld inter- specific backcross mice. Black boxes represent the homozygous C3H pattern, and white boxes represent the FI pattern.

leukin-la (ILla) is shown in Fig. 6. The best gene order & S.D. indicated that Thbsl was located 13.2 f 3.3 cM distal to glucagon and 2.6 & 1.5 cM proximal to 11-la. This localization confirmed the results of a previous study that mapped Thbsl to region F of mouse chromosome 2 using in situ hybridization (62).

DISCUSSION

The recent discovery of a second expressed mouse TSP gene prompted this characterization of the mTSPl and mTSP2 gene products based on their predicted amino acid sequences and expression patterns. The cloning and charac- terization of full-length mTSP1 and mTSP2 allowed, for the first time, a sequence comparison with the previously cloned human TSP and the more recently cloned chicken TSP. The predicted amino acid sequences of hTSP, mTSP1, mTSP2, and chTSP are highly conserved, particularly at the carboxyl terminus of the molecules. However, significant differences at the amino terminus of the molecules allowed the identifi- cation of hTSP as a TSPl homolog and chTSP as a TSPZ homolog. The higher degree of homology between TSPl or TSP2 molecules of different species than between the two different mouse TSP molecules suggests that the ancestral TSP gene duplicated and evolved prior to the divergence of birds and mammals and also predicts the existence of a chicken TSPl homolog.

A number of amino acid residues and functional domains known to be critical for TSP structure are conserved among the various TSPs, suggesting that TSPl and TSPZ molecules have very similar secondary, tertiary, and quaternary struc- tures. The conservation of the 2 cysteines involved in inter- chain disulfide linkage and trimer assembly is evidence that TSP2, like TSP1, probably forms trimers. Supporting the likelihood of TSPZ trimer formation is the conservation of cysteines in the cysteine-rich procollagen domain, which may

3280 Mouse Thrombospondin 2 Sequence and Expression

facilitate oligomer assembly (42). The interesting question of whether TSPl and TSP2 molecules exist exclusively as hom- otrimers or are able to form heterotrimers remains to be answered. All other cysteines, except the free reactive sulfhy- dryl of TSP1, are also conserved among the various TSPs. Since these cysteines are presumably involved in the intra- chain and interchain disulfide linkages that determine TSP structure, their absolute conservation indicates a similar structure for TSPl and TSP2. The regions of TSPl and TSP2 containing type 3 repeats also appear to be structurally similar since the aspartic acid residues that participate in the binding of calcium are conserved. The strong conservation of TSP2 domains known to determine TSPl structure and me- diate its interactions with a variety of ligands suggests that TSPZ may also be able to interact with these ligands in a similar manner and thus share some functional features with TSP1.

TSPl’s cell adhesive properties are mediated primarily by distinct domains. Interestingly, these adhesive domains are significantly different in TSP2. The conservation of positively charged amino acids in the two cationic amino acid clusters of the heparin-binding domain suggests that both TSPl and TSPZ bind heparin; but since mTSP2 and chTSP2 do not contain the full complement of charged residues, their heparin and heparan sulfate proteoglycan binding affinities may be lower. The VTCG sequence located in TSP type 1 repeats is highly conserved in type 1 homology repeats found in other proteins including thrombospondin-related anonymous pro- tein of P. falciparum and circumsporozite protein of P. vivax (44). It was recently shown that the VTCG sequence is critical for the cell adhesive properties of peptides based on the circumsporozite protein sequence (44); similarly, TSP pep- tides containing VTCG serve as attachment factors for mel- anoma cells (63). TSPl contains two VTCG sequences, and TSP2 contains three VTCG sequences. However, in mTSP2, the glycine in the third VTCG sequence is replaced by an alanine; it is not known if this conservative substitution or the presence of an additional VTCG sequence in TSP2 changes adhesive properties. The RGDA sequence in the carboxyl-terminal domain of hTSP is recognized by integrin receptor and may, in part, mediate the attachment of endothelial cells to TSP (46). What effect the conservative substitution of an isoleucine for the alanine in mTSP2 and chTSP has on adhesion remains to be determined. The effects of amino acid substitutions in adhesion domains on the cell adhesive properties of TSPZ remain to be determined. How- ever, it can be predicted that alterations in the adhesive properties of TSPS could potentially modulate the function of TSP2 in processes involving cell adhesion such as cell migration, inflammation, and embryogenesis.

Human TSP, the homolog of mTSP1, has been demon- strated to be a primary or immediate-early respone gene to serum and other growth factors including PDGF, FGF, and IL-lp (12). Mouse TSPl has also been shown to be a primary response gene to FGF in cells in which FGF triggers a mito- genic response (11). In this study, the distinct patterns of mTSPl and mTSP2 expression were examined to identify the individual roles of each molecule in the related processes of cell growth and embryogenesis. The rapid induction of mTSP2 in the absence of new protein synthesis demonstrates that mTSP2, like mTSP1, is a primary response gene to a variety of growth-promoting stimuli including serum, basic FGF, PDGF, and IL-1p. The induction of mTSPl and mTSP2 mRNA levels by cyloheximide implies that mTSPl and mTSP2 mRNA levels are regulated by a labile ribonuclease or transcriptional repressor. The 2166-bp 3’-untranslated re-

gion of the hTSPl gene contains 37 TATT or ATTT(A) motifs identified in other genes as mediators of rapid mRNA turnover (64). Within the 370 bp of mTSP2 3’-untranslated region sequenced to date, nine such motifs exist, suggesting that mRNA stabilization may be a mechanism regulating mTSP2 mRNA expression.

Thrombospondin, like other extracellular matrix proteins, has been shown to have distinct regional and temporal pat- terns of expression during development, its presence generally associated with areas of cellular proliferation and migration and tissue remodeling (16, 17). In fact, it has been hypothe- sized that TSP, by binding both plasminogen and tissue plasminogen activator and thus enhancing plasmin genera- tion, forms a nidus for the generation of proteases critical for cell migration and tissue remodeling (65). From this study, it is apparent that TSPl and TSPP display some distinct tissue- and stage-specific expression patterns, suggesting that differ- ential, or perhaps combinatorial, expression of TSP has a role in normal mammalian development. Although both TSPl and TSP2 are expressed at high levels during organogenesis and fetal development of the lung and kidney, TSPS expression is much higher relative to TSPl expression in the brain during these developmental stages, possibly reflecting a difference in the particular cell types associated with each organ. The expression of TSP genes appears to be temporally regulated during neurulation and early organogenesis. For example, TSP2 mRNA is not detected in either the head or body until day 13, whereas TSPl is detected at the earliest stage exam- ined (day 11). The significance of the large increase in both TSPl and TSPZ mRNA levels in the heart following birth may be related to the switch from low pressure maternal circulation to the higher pressure post-birth circulation that requires a degree of cardiac hypertrophy. The discrete and transient expression of mTSPl seen in day 15 embryonic liver may represent a role for mTSPl in the proliferation of fetal liver hematopoietic cells that accompanies the switch of the site of hematopoiesis from the yolk sac to the fetal liver at approximately day 13. Consistent with this hypothesis, TSP has been shown to be a potent attachment factor for hema- topoietic progenitor cells of all three lineages (erythrocyte, megakaryocyte, and granulocyte) (66). The general tissue- specific TSP mRNA expression patterns seen in this study correlate with TSP protein expression patterns seen in pre- vious studies (17). However, it will be necessary to examine the discrete patterns of mTSPl and mTSP2 mRNA and protein expression by immunocytochemistry and in situ hy- bridization procedures using reagents that specifically recog- nize mTSP1 or mTSP2 to determine the cell populations associated with expression of each TSP molecule.

The localization of the mTSPl gene (Thbsl ) to the central portion of mouse chromosome 2 confirms the localization previously performed (62). The localization of the mTSP2 gene (Thbs2) to a different chromosome (chromosome 17) provides unequivocal evidence for the existence of two TSP genes. Thbs2 lies between the t haplotype locus and the H2 major histocompatibility locus on chromosome 17. Interest- ingly, these loci have previously been associated with mouse development and immunological function, respectively (41).

From this study, it is apparent that mTSP2, like mTSP1, is expressed during cell proliferation and embryogenesis, pre- sumably functioning through many of the same structural and adhesive domains identified in hTSP1. The precise effects of structural differences and the differential regulation of mTSPl and mTSP2 gene expression during mitogeneis and development will be important areas of future investigation.

Acknowledgments-We thank the following individuals for their

Mouse Thrombospondin 2

kind gifts of probes used in the chromosomal localization studies: Drs. G. I. Bell for pshglul, L. M. Silver for pTcrlO, S. B. Mizel for ~1111301, and H. C. Passmore for pE. We also thank Thad A. Howard for expert technical assistance and Monica Zepeda for her contribu- tion to the cloning of mTSP1. In addition, we are indebted to Drs. Rachel Yabkowitz and Vidya Sarma for critical readings of the manuscript.

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