Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor.

Growth of tumors and metastasis are processes known to require neovascularization. To ascertain the participation of the endogenous angiogenic inhibitor thrombospondin-1 (TSP1) in tumor progression, we generated mammary tumor-prone mice that either lack, or specifically overexpress, TSP1 in the mammary gland. Tumor burden and vasculature were significantly increased in TSP1-deficient animals, and capillaries within the tumor appeared distended and sinusoidal. In contrast, TSP1 overexpressors showed delayed tumor growth or lacked frank tumor development (20% of animals); tumor capillaries showed reduced diameter and were less frequent. Interestingly, absence of TSP1 resulted in increased association of vascular endothelial growth factor (VEGF) with its receptor VEGFR2 and higher levels of active matrix metalloproteinase-9 (MMP9), a molecule previously shown to facilitate both angiogenesis and tumor invasion. In vitro, enzymatic activation of proMMP9 was suppressed by TSP1. Together these results argue for a protective role of endogenous inhibitors of angiogenesis in tumor growth and implicate TSP1 in the in vivo regulation of metalloproteinase-9 activation and VEGF signaling.

Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice.

The cyclooxygenase (COX)-2 gene encodes an inducible prostaglandin synthase enzyme that is overexpressed in adenocarcinomas and other tumors. Deletion of the murine Cox-2 gene in Min mice reduced the incidence of intestinal tumors, suggesting that it is required for tumorigenesis. However, it is not known if overexpression of Cox-2 is sufficient to induce tumorigenic transformation. We have derived transgenic mice that overexpress the human COX-2 gene in the mammary glands using the murine mammary tumor virus promoter. The human Cox-2 mRNA and protein are expressed in mammary glands of female transgenic mice and were strongly induced during pregnancy and lactation. Female virgin Cox-2 transgenic mice showed precocious lobuloalveolar differentiation and enhanced expression of the beta-casein gene, which was inhibited by the Cox inhibitor indomethacin. Mammary gland involution was delayed in Cox-2 transgenic mice with a decrease in apoptotic index of mammary epithelial cells. Multiparous but not virgin females exhibited a greatly exaggerated incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors. Cox-2-induced tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-x(L) and an increase in the anti-apoptotic protein Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2 expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.

Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice.

We have generated transgenic mice that harbor a 140 kb genomic fragment of the human BRCA1 locus (TgN x BRCA1GEN). We find that the transgene directs appropriate expression of human BRCA1 transcripts in multiple mouse tissues, and that human BRCA1 protein is expressed and stabilized following exposure to DNA damage. Such mice are completely normal, with no overt signs of BRCA1 toxicity commonly observed when BRCA1 is expressed from heterologous promoters. Most importantly, however, the transgene rescues the otherwise lethal phenotype associated with the targeted hypomorphic allele (Brca1DeltaexllSA). Brca1-/-; TgN x BRCA1GEN bigenic animals develop normally and can be maintained as a distinct line. These results show that a 140 kb fragment of chromosome 17 contains all elements necessary for the correct expression, localization, and function of the BRCA1 protein. Further, the model provides evidence that function and regulation of the human BRCA1 gene can be studied and manipulated in a genetically tractable mammalian system. Oncogene (2000) 19, 4085 - 4090

Regulation of VEGF and VEGF receptor expression in the rodent mammary gland during pregnancy, lactation, and involution.

Vascular endothelial growth factors (VEGFs) are endothelial cell-specific mitogens with potent angiogenic and vascular permeability-inducing properties. VEGF, VEGF-C, and VEGFRs -1, -2, and -3 were found to be expressed in post-pubertal (virgin) rodent mammary glands. VEGF was increased during pregnancy (5-fold) and lactation (15-19-fold). VEGF-C was moderately increased during pregnancy and lactation (2- and 3-fold respectively). VEGF levels were reduced by approximately 75% in cleared mouse mammary glands devoid of epithelial components, demonstrating that although the epithelial component is the major source of VEGF, approximately 25% is derived from stroma. This was confirmed by the findings (a) that VEGF transcripts were expressed predominantly in ductal and alveolar epithelial cells, and (b) that VEGF protein was localized to ductal epithelial cells as well as to the stromal compartment including vascular structures. VEGF was detected in human milk. Finally, transcripts for VEGFRs -2 and -3 were increased 2-3-fold during pregnancy, VEGFRs -1, -2 and -3 were increased 2-4-fold during lactation, and VEGFRs -2 and -3 were decreased by 20-50% during involution. These results point to a causal role for the VEGF ligand-receptor pairs in pregnancy-associated angiogenesis in the mammary gland, and suggest that they may also regulate vascular permeability during lactation.

METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity.

We have studied two related proteins that contain a repeated amino acid motif homologous to the anti-angiogenic type 1 repeats of thrombospondin-1 (TSP1). Complete sequence analysis revealed no other similarities with TSP1, but identified unique signal sequences, as well as metalloprotease and disintegrin-like domains in the NH(2) termini. We named these proteins METH-1 and METH-2 due to the novel combination of metalloprotease and thrombospondin domains. Overall amino acid sequence identity between METH-1 and METH-2 is 51. 7%, yet transcript distribution revealed non-overlapping patterns of expression in tissues and cultured cell lines. To characterize these proteins functionally, we isolated full-length cDNAs, produced recombinant protein, and generated antisera to the recombinant proteins. Both METH-1 and METH-2 represent single copy genes, which encode secreted and proteolytically processed proteins. METH proteins suppressed fibroblast growth factor-2-induced vascularization in the cornea pocket assay and inhibited vascular endothelial growth factor-induced angiogenesis in the chorioallantoic membrane assay. Suppression of vessel growth in both assays was considerably greater than that mediated by either thrombospondin-1 or endostatin on a molar basis. Consistent with an endothelial specific response, METH-1 and METH-2 were shown to inhibit endothelial cell proliferation, but not fibroblast or smooth muscle growth. We propose that METH-1 and METH-2 represent a new family of proteins with metalloprotease, disintegrin, and thrombospondin domains. The distinct distribution of each gene product suggests that each has evolved distinct regulatory mechanisms that potentially allow for fine control of activity during distinct physiological and pathological states.

Wnt-10b directs hypermorphic development and transformation in mammary glands of male and female mice.

Wnt-10b is expressed during the formation of the mammary rudiment in mouse embryos and its expression continues through puberty when the mammary ductal pattern is established under control of ovarian steroids. Recently, viral activation of the Wnt-10b locus has linked its overexpression to mammary tumor formation, suggesting a role for Wnt-10b in patterning and growth-regulation of the mammary gland. To test this notion, we created lines of transgenic mice that express elevated levels of Wnt-10b under the control of the MMTV promoter/enhancer. Overexpression of this gene resulted in profound developmental alterations in the mammary gland, including expanded glandular development and the precocious appearance of alveoli in virgin females. Moreover, transgenic male mice also exhibited dramatic mammary development involving highly branched mammary ducts and gynecomastia. Aberrant expression of Wnt-10b in the mammary rudiments of males evidently bypasses the normal requirement for ovarian hormonal control in stimulating mammary ductal growth and the repressive effects of androgens. In addition to these developmental effects, transgenic mice of both sexes were highly susceptible to the development of mammary adenocarcinomas. Such tumors arose in a solitary manner indicating that Wnt-10b is a proto-oncogene which provides a necessary, but insufficient signal for oncogenesis. Relevant to this, there was no evidence of amplified expression of FGF mRNAs in these tumors though the Fgf's are a class of genes often implicated as collaborators in Wnt-mediated tumor formation. Indeed, co-expression of MMTV-Wnt-10b and MMTV-FGF-3/int-2 resulted in sterile offspring with highly disorganized mammary epithelium, demonstrating a potent interaction between their respective developmental pathways. These results suggest that Wnt-10b, or other Wnt genes expressed early in mammary development, play a role in regulating sexual dimorphism and show potent transforming activity when overexpressed.

Expression of Brca1 is associated with terminal differentiation of ectodermally and mesodermally derived tissues in mice.

We have isolated genomic and cDNA clones of Brca1, a mouse homolog of the recently cloned breast cancer-associated gene, BRCA1. Brca1 encodes an 1812-amino-acid protein with a conserved zinc finger domain and significant homology to the human protein. Brca1 maps to Chromosome 11 within a region of conserved synteny with human chromosome 17, consistent with the mapping of the human gene to 17q21. Brca1 transcripts are expressed in a variety of cultured cells but reveal a specific and dynamic expression pattern during embryonic development. For example, expression is observed first in the otic vesicle of embryonic day 9.5 (E9.5) embryos. This expression diminishes and is replaced by expression in the neuroectoderm at E10.5. By E11-12.5, higher levels are observed in differentiating keratinocytes and in whisker pad primordia. Transcripts also become evident in epithelial cells of the E14-17 kidney. Brca1 expression occurs in differentiating epithelial cells of several adult organs as well, suggesting a general role in the functional maturation of these tissues. Consistent with this, Brca1 transcripts are expressed in both alveolar and ductal epithelial cells of the mammary gland. During pregnancy, there is a large increase in Brca1 mRNA in mammary epithelial cells, an increase that parallels their functional differentiation. Because high rates of breast cancer are associated with loss of BRCA1 in humans, it is possible that this gene provides an important growth regulatory function in mammary epithelial cells. In addition, increased transcription of mammary Brca1 during pregnancy might contribute, in part, to the reduced cancer risk associated with exposure to pregnancy and lactation.

Distribution of SPARC in normal and neoplastic human tissue.

SPARC (Secreted Protein, Acidic and Rich in Cysteine)/osteonectin is a secreted glycoprotein that exhibits restricted expression in murine adult and embryonic tissues and is associated with cell migration, matrix mineralization, steroid hormone production, cell cycle regulation, and angiogenesis. We produced a monoclonal antibody, MAb SSP2, against a Ca(2+)-binding region of SPARC and evaluated the immunoreactivity of normal and malignant tissue from 118 human samples. In normal tissue we found restricted and moderate reactivity with SSP2 in steroidogenic cells, chondrocytes, placental trophoblasts, vascular smooth muscle cells, and endothelial cells. Strong reactivity was found in fibrocytes and endothelial cells involved in tissue repair and in invasive malignant tumors, including those of the gastrointestinal tract, breast, lung, kidney, adrenal cortex, ovary, and brain. We conclude that SSP2 is a useful reagent for detection of SPARC in human tissue. Given the broad reactivity of malignant tissues, we propose that SPARC expression might contribute to some aspects of tumor progression.

Insertional mutagenesis identifies a member of the Wnt gene family as a candidate oncogene in the mammary epithelium of int-2/Fgf-3 transgenic mice.

Transgenic mice harboring the int-2/Fgf-3 protooncogene under transcriptional control of the mouse mammary tumor virus (MMTV) promoter/enhancer exhibit a dramatic, benign hyperplasia of the mammary gland. In one int-2 transgenic line (TG.NX), this growth disturbance is evoked by pregnancy and regresses after parturition. Regression of hyperplastic mammary epithelium is less complete after successive pregnancies, and, within 10 months, most TG.NX mice stochastically develop mammary carcinomas that are transplantable in virgin, syngeneic mice. To identify genes that cooperate with int-2 in cell transformation, we infected TG.NX transgenic mice with MMTV. In a cohort of 14 animals, most mammary tumors represented clonal or oligoclonal outgrowths harboring one to five proviral MMTV integrants. Eight of 35 (23%) MMTV+ tumors exhibited proviral insertion at the Wnt-1 locus. No provirus was detected at the int-2, int-3, or Wnt-3 loci. By Southern analysis, two tumors had proviral insertions at the same genomic location, which was mapped to chromosome 15. Cloning of this int locus identified an additional member of the Wnt gene family. The predicted 389-amino acid protein is most closely related to zebrafish Wnt-10a (58% amino acid identity over 362 residues) and, based on homology analysis, was designated Wnt-10b. This newly discovered Wnt family member was expressed in the embryo and mammary gland of virgin but not pregnant mice and represents a candidate collaborating oncogene of int-2/Fgf-3 in the mammary epithelium.

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.

SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin-like region and the Ca(2+)-binding EF-hand.

SPARC is a one of a group of extracellular matrix proteins that regulate cell adhesion through a loss of focal adhesion plaques from spread cells. We previously reported that SPARC reduced the number of bovine aortic endothelial (BAE) cells positive for focal adhesions [Murphy-Ullrich et al. (1991): J Cell Biol 115:1127-1136]. We have now characterized the effect of SPARC on the cytoskeleton of BAE cells. Addition of SPARC to spread BAE cells caused a dose-dependent loss of focal adhesion-positive cells, that was maximal at approximately 1 microgram/ml (0.03 microM). Consistent with the loss of adhesion plaques as detected by interference reflection microscopy, vinculin appeared diffuse and F-actin was redistributed to the periphery of cells incubated with SPARC. However, the distribution of the integrin alpha v beta 3 remained clustered in a plaque-like distribution. These data, and the observation that SPARC binds to BAE cells but not to the extracellular matrix, indicate that SPARC acts via interactions with cell surface molecules and not by steric/physical disruption of integrin-extracellular matrix ligands. To determine the region(s) of SPARC that mediate a loss of focal adhesions, we tested peptides from the four distinct regions of SPARC. The cationic, cysteine-rich peptide 2.1 (amino acids 54-73) and the Ca(2+)-binding EF-hand-containing peptide 4.2 (amino acids 254-273) were active in focal adhesion disassembly. Furthermore, antibodies specific for these regions neutralized the focal adhesion-labilizing activity of SPARC. These results are consistent with previous data showing that peptide 2.1 and 4.2 interact with BAE cell surface proteins and indicate that the loss of focal adhesions from endothelial cells exposed to SPARC is a receptor-mediated event.

Inhibition of endothelial cell proliferation by SPARC is mediated through a Ca(2+)-binding EF-hand sequence.

SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin and BM-40) is a metal-binding glycoprotein secreted by a variety of cultured cells and characteristic of tissues undergoing morphogenesis, remodeling, and repair. Recently it has been shown that SPARC inhibits the progression of the endothelial cell cycle in mid-G1, and that a synthetic peptide (amino acids 54-73 of secreted murine SPARC, peptide 2.1) from a cationic, disulfide-bonded region was in part responsible for the growth-suppressing activity [Funk and Sage (1991): Proc Natl Acad Sci USA 88:2648-2652]. Moreover, SPARC was shown to interact directly with bovine aortic endothelial (BAE) cells through a C-terminal EF-hand sequence comprising a high-affinity Ca(2+)-binding site of SPARC and represented by a synthetic peptide (amino acids 254-273) termed 4.2 [Yost and Sage (1993): J Biol Chem 268:25790-25796]. In this study we show that peptide 4.2 is a more potent inhibitor of DNA synthesis that acts cooperatively with peptide 2.1 to diminish the incorporation of [3H]-thymidine by both BAE and bovine capillary endothelial (BCE) cells. At concentrations of 0.019-0.26 mM peptide 4.2, thymidine incorporation by BAE cells was decreased incrementally, relative to control values, from approximately 100 to 10%. Although somewhat less responsive, BCE cells exhibited a dose-responsive decrement in thymidine incorporation, with a maximal inhibition of 55% at 0.39 mM. The inhibitory effect of peptide 4.2 was essentially independent of heparin and basic fibroblast growth factor and was blocked by anti-SPARC peptide 4.2 IgG, but not by antibodies specific for other domains of SPARC. To identify residues that were necessary for inhibition of DNA synthesis, we introduced single amino acid substitutions into synthetic peptide 4.2 and tested their activities and cell-surface binding characteristics on endothelial cells. Two peptides displayed null to diminished effects in the bioassays that were concentration-dependent: peptide 4.2 K, containing an Asp258 --> Lys substitution, and peptide 4.2 AA, in which the two disulfide-bonded Cys (positions 255 and 271) were changed to Ala residues. Peptide 4.2 K, which failed to fulfill the EF-hand consensus formula, exhibited an anomalous fluorescence emission spectrum, in comparison with the wild-type 4.2 sequence, that was indicative of a compromised affinity for Ca2(+).(ABSTRACT TRUNCATED AT 400 WORDS)

SPARC is a source of copper-binding peptides that stimulate angiogenesis.

SPARC is a transiently expressed extracellular matrix-binding protein that alters cell shape and regulates endothelial cell proliferation in vitro. In this study, we show that SPARC mRNA and protein are synthesized by endothelial cells during angiogenesis in vivo. SPARC and peptides derived from a cationic region of the protein (amino acids 113-130) stimulated the formation of endothelial cords in vitro; moreover, these peptides stimulated angiogenesis in vivo. Mapping of the active domain demonstrated that the sequence KGHK was responsible for most of the angiogenic activity; substitution of the His residue decreased the effect. We found that proteolysis of SPARC provided a source of KGHK, GHK, and longer peptides that contained these sequences. Although the Cu(2+)-GHK complex had been identified as a mitogen/morphogen in normal human plasma, we found KGHK and longer peptides to be potent stimulators of angiogenesis. SPARC113-130 and KGHK were shown to bind Cu2+ with high affinity; however, previous incubation with Cu2+ was not required for the stimulatory activity. Since a peptide from a second cationic region of SPARC (SPARC54-73) also bound Cu2+ but had no effect on angiogenesis, the angiogenic activity appeared to be sequence specific and independent of bound Cu2+. Thus, specific degradation of SPARC, a matrix-associated protein expressed by endothelial cells during vascular remodeling, releases a bioactive peptide or peptides, containing the sequence (K)GHK, that could regulate angiogenesis in vivo.

The biology of SPARC, a protein that modulates cell-matrix interactions.

An extracellular matrix-associated glycoprotein expressed in a variety of tissues during embryogenesis and repair, SPARC contains modular domains that can function independently to bind cells and matrix components. Because SPARC can selectively disrupt cellular contacts with matrix and thereby effect changes in cell shape, it has been referred to as an antiadhesin. Inhibition of the expression of SPARC altered axial development in frogs, and deregulated expression in nematode worms resulted in a derangement of muscle attachment and embryonic lethality. SPARC also inhibits cell cycle progression in vitro, in part through a cationic, disulfide-bonded region that is homologous to a repeated domain in the cytokine inhibitor, follistatin. Moreover, SPARC binds specifically to the B chain of platelet-derived growth factor and alters the response of cells to several cytokines. Although information concerning the expression, biochemical properties, and cellular activities of SPARC has increased significantly over the last decade, the precise function of the protein has not been resolved. Goals of future studies include characterization of cell-surface receptors for SPARC and the interactions with morphogens and growth factors that regulate specific activities during animal development.

Molecular analysis of chicken embryo SPARC (osteonectin).

SPARC is a secreted glycoprotein that modulates cell shape and cell-matrix interactions. Levels of SPARC are increased at sites of somitogenesis, osteogenesis, and angiogenesis in the embryo and during wound repair in the adult. We have cloned and characterized SPARC from chicken embryo. A 2.2-kbp cDNA, obtained by a novel use of the polymerase chain reaction, was determined to encode a 298-residue protein that is 85% identical to human SPARC. Antigenic sites in particular appear to be highly conserved, as antibodies against C-terminal sequences of murine and bovine SPARC reacted with a 41-43 kDa protein in chicken embryo extracts. Chicken SPARC can be defined by four sequence signatures: (a) a conserved spacing of 11 cysteine residues in domain II, (b) the pentapeptide KKGHK in domain II, which is contained within a larger region of 31 identical residues, (c) a 100% conserved region of 10 residues in domain III, and (d) a C-terminal, calcium-binding EF-hand motif. SPARC mRNAs in the 10-day-old chicken embryo are represented by three sizes of 1.8, 2.2 and 3.0 kb. The relative steady-state levels for the 2.2-kb mRNA were determined as aorta > or = skeletal muscle > calvarium > vertebra > anterior limb > kidney > heart > brain > skin and lung >> liver. The relative abundance of the 1.8-kb and 2.2-kb mRNAs varied among tissues and indicated that differential processing of SPARC mRNAs might occur. All three RNA species were detected by a cDNA probe for the N-terminal part of the coding region. Thus, the three mRNA species appear to arise from differential 3' splicing and/or polyadenylation. Collective evidence demonstrates that SPARC has been well-conserved during vertebrate evolution, a finding that indicates a fundamental role for this protein in development.

Differential expression of SPARC and thrombospondin 1 in wound repair: immunolocalization and in situ hybridization.

SPARC and thrombospondin 1 (TSP-1) are secreted glycoproteins expressed by similar types of cells in culture and in tissues. To compare these two proteins in vivo, we analyzed the differential expression of SPARC and TSP-1 during wound repair. Full-thickness incision wounds were made in rats and biopsied at 12 hr-14 days. Antibodies against SPARC revealed an increased proportion of immunoreactive fibroblastic cells at the wound edge at 3 days with maximal numbers at 7 days. In situ hybridization for SPARC produced results consistent with those of immunohistochemistry. With combined immunohistochemistry and in situ hybridization, some of the macrophages at the wound edge expressed SPARC mRNA. In contrast, immunoreactivity for TSP-1 was extracellular; expression at the wound edge was noted at 12 hr and was maximal at 1-2 days. TSP-1 mRNA was found in the thrombus, but not at the wound edge. In conclusion, SPARC and TSP-1 have contrasting roles during wound healing. SPARC expression from the middle through late stages of repair was consistent with its previously proposed functions in remodeling; in contrast, the transient expression of TSP-1 early in repair might facilitate the action of other proteins in recruitment and/or proliferation of cells in the healing wound.

SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway.

SPARC (osteonectin/BM40) is a secreted protein that modifies the interaction of cells with extracellular matrix (ECM). When we added SPARC to cultured rabbit synovial fibroblasts and analyzed the secreted proteins, we observed an increase in the expression of three metalloproteinases--collagenase, stromelysin, and the 92-kD gelatinase--that together can degrade both interstitial and basement membrane matrices. We further characterized the regulation of one of these metalloproteinases, collagenase, and showed that both collagenase mRNA and protein are upregulated in fibroblasts treated with SPARC. Experiments with synthetic SPARC peptides indicated that a region in the neutral alpha-helical domain III of the SPARC molecule, which previously had no described function, was involved in the regulation of collagenase expression by SPARC. A sequence in the carboxyl-terminal Ca(2+)-binding domain IV exhibited similar activity, but to a lesser extent. SPARC induced collagenase expression in cells plated on collagen types I, II, III, and V, and vitronectin, but not on collagen type IV. SPARC also increased collagenase expression in fibroblasts plated on ECM produced by smooth muscle cells, but not in fibroblasts plated on a basement membrane-like ECM from Engelbreth-Holm-Swarm sarcoma. Collagenase was induced within 4 h in cells treated with phorbol diesters or plated on fibronectin fragments, but was induced after 8 h in cells treated with SPARC. A number of proteins were transiently secreted by SPARC-treated cells within 6 h of treatment. Conditioned medium that was harvested from cultures 7 h after the addition of SPARC, and depleted of residual SPARC, induced collagenase expression in untreated fibroblasts; thus, part of the regulation of collagenase expression by SPARC appears to be indirect and proceeds through a secreted intermediate. Because the interactions of cells with ECM play an important role in regulation of cell behavior and tissue morphogenesis, these results suggest that molecules like SPARC are important in modulating tissue remodeling and cell-ECM interactions.

Regulation of gene expression by SPARC during angiogenesis in vitro. Changes in fibronectin, thrombospondin-1, and plasminogen activator inhibitor-1.

Angiogenesis in vitro, the formation of capillary-like structures by cultured endothelial cells, is associated with changes in the expression of several extracellular matrix proteins. The expression of SPARC, a secreted collagen-binding glycoprotein, has been shown to increase significantly during this process. We now show that addition of purified SPARC protein, or an N-terminal synthetic peptide (SPARC4-23), to strains of bovine aortic endothelial cells undergoing angiogenesis in vitro resulted in a dose-dependent decrease in the synthesis of fibronectin and thrombospondin-1 and an increase in the synthesis of type 1-plasminogen activator inhibitor. SPARC decreased fibronectin mRNA by 75% over 48 h, an effect that was inhibited by anti-SPARC immunoglobulins. Levels of thrombospondin-1 mRNA were diminished by 80%. Over a similar time course, both mRNA and protein levels of type 1-plasminogen activator inhibitor (PAI-1) were enhanced by SPARC and the SPARC4-23 peptide. The effects were dose-dependent with concentrations of SPARC between 1 and 30 micrograms/ml. In contrast, no changes were observed in the levels of either type I collagen mRNA or secreted gelatinases. Half-maximal induction of PAI-1 mRNA or inhibition of fibronectin and thrombospondin mRNAs occurred with 2-5 micrograms/ml SPARC and approximately 0.05 mM SPARC4-23. Strains of endothelial cells that did not form cords and tubes in vitro had reduced or undetectable responses to SPARC under identical conditions. These results demonstrate that SPARC modulates the synthesis of a subset of secreted proteins and identify an N-terminal acidic sequence as a region of the protein that provides an active site. SPARC might therefore function, in part, to achieve an optimal ratio among different components of the extracellular matrix. This activity would be consistent with known effects of SPARC on cellular morphology and proliferation that might contribute to the regulation of angiogenesis in vivo.

Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro.

Vascular endothelial cells that are cultured on layers of gelled basement membrane matrix organize rapidly into networks of cords or tubelike structures. Although this phenomenon is a potential model for angiogenesis in vivo, we questioned whether basement membrane matrix directs the differentiation of endothelial cells in a specific manner. In this study, we have examined factors that influence the formation of cellular networks in vitro in an attempt to define a basic mechanism for this process. We found that endothelial cells, fibroblasts, smooth muscle cells, and cells of the murine Leydig cell line TM3 formed networks on basement membrane matrix in much the same fashion. Light and electron microscopy, combined with time-lapse videomicroscopy, revealed that cells organized on a tesselated network of aligned basement membrane matrix that was generated by tension forces of cellular traction. Cellular elongation and progressive motility across the surface of the gel were restricted to tracks of aligned matrix and did not occur until the tracks appeared. The formation of cellular networks on basement membrane matrix was inhibited by reducing the thickness of the matrix, by including native type I collagen in the matrix, or by disrupting cytoskeletal microfilaments and microtubules. Cell division was not required for network formation. Bovine aortic endothelial cells that formed networks did not simultaneously transcribe mRNA for type I collagen, a protein synthesized by endothelial cells that form tubes spontaneously in vitro. Moreover, levels of mRNA for fibronectin and SPARC (Secreted Protein that is Acidic and Rich in Cysteine) in network-forming cells were similar to levels seen in endothelial cells that did not form networks. Endothelial cells and TM3 cells that were plated on highly malleable gels of native type I collagen also formed cords and aligned matrix fibers into linear tracks that resembled those generated on basement membrane matrix, although the structures were not as well-defined. Our observations suggest that the mechanochemical properties of extracellular matrices are able to translate the forces of cellular traction into templates that direct the formation of complex cellular patterns.

The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors.

Interactions among growth factors, cells, and extracellular matrix are critical to the regulation of directed cell migration and proliferation associated with development, wound healing, and pathologic processes. Here we report the association of PDGF-AB and -BB, but not PDGF-AA, with the extracellular glycoprotein SPARC. Complexes of SPARC and 125I-labeled PDGF-BB or -AB were specifically immunoprecipitated by anti-SPARC immunoglobulins. 125I-PDGF-BB and -AB also bound specifically to SPARC that was immobilized on microtiter wells or bound to nitrocellulose after transfer from SDS/polyacrylamide gels. The binding of PDGF-BB to SPARC was pH-dependent; significant binding was detectable only above pH 6.6. The interaction of SPARC with specific dimeric forms of PDGF affected the activity of this mitogen. SPARC inhibited the binding of PDGF-BB and PDGF-AB, but not PDGF-AA, to human dermal fibroblasts in a dose-dependent manner. The expression of SPARC and PDGF was minimal in most normal adult tissues but was increased after injury. Enhanced expression of both PDGF-B chain and SPARC was seen in advanced lesions of atherosclerosis. We suggest that the coordinate expression of SPARC and PDGF-B-containing dimers following vascular injury may regulate the activity of specific dimeric forms of PDGF in vivo.