Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing, and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was removed from perlecan by heparitinase treatment, and the ϳ450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of ϳ300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin, rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of the growth factor.

Perlecan is a ubiquitous pericellular proteoglycan ideally placed to mediate cell signaling events controlling migration, proliferation and differentiation. Its control of growth factor signaling usually involves interactions with the heparan sulfate chains covalently coupled to the protein core's Nterminus. However, this modular protein core also binds with relatively high affinity to a number of growth factors and surface receptors, thereby stabilizing cell-matrix links. This review will focus on perlecan/growth factor interactions and describe recent advances in our understanding of this highlyconserved proteoglycan during development, cancer growth and angiogenesis. The pro-angiogenic capacities of perlecan that involve proliferative and migratory signals in response to bound growth factors will be explored, as well as the anti-angiogenic signals resulting from interactions between the C-terminal domain known as endorepellin and integrins that control cell adhesion to the extracellular matrix. These two somewhat diametrically-opposed roles will be discussed in light of new data emerging from various fields which converge on perlecan as a key regulator of cell growth and angiogenesis.Perlecan was originally isolated in 1980 by Hassell and coworkers from the Engelbreth-HolmSwarm sarcoma, a basement membrane-secreting tumor, and was soon demonstrated to be expressed also at the cell surface of human colon carcinoma cells (1). In spite of their differential expression, the two molecules were shown to have biosynthetic and immunological similarities. Because of its large size -the mRNA encoding perlecan is ~15 kb-it took over a decade of efforts to complete the cDNA cloning of the full-length mouse perlecan, followed by the complete structure of the human counterpart, its chromosomal mapping, and its genomic organization (2). The eponym "perlecan" derives from its ultrastructural appearance of "beads on a string", a feature attributable to the various globular domains interspersed among more linear structures (1).Perlecan is composed of five distinct domains with homology to growth factors and to protein modules involved in lipid metabolism, cell adhesion, and homotypic and heterotypic interactions (3). Notably, the N-terminal domain I contains three attachment sites for the heparan sulfate side chains with additional attachment sites in the carboxyl terminus domain V (Figure 1). Interestingly, the other two main HSPGs from basement membranes, collagen XVIII and agrin, do not share much structural homology with exception of agrin domain V. Collagen XVIII is a member of the subfamily of collagens, also known as multiplexins. These † This work was supported in part by NIH grants RO1 CA39481, RO1 CA47282, and RO1 CA120975 (R.V.I.), NH&MRC Project Grant 512167 (J.M. & J.W.) and ARC Discovery Project Grant DP0557863 & ARC Linkage Grants LP0455407 and LX0667295 (J.W.) *To whom correspondence should be addressed. Phone: 215-503-2208. Fax: 215-923-7969. E-mail: iozzo@mail.jci collagens, which include collagen XV,...

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Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycyclineinducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In

Smooth muscle cell proliferation can be inhibited by heparan sulfate proteoglycans whereas the removal or digestion of heparan sulfate from perlecan promotes their proliferation. In this study we characterized the glycosaminoglycan side chains of perlecan isolated from either primary human coronary artery smooth muscle or endothelial cells and determined their roles in mediating cell adhesion and proliferation, and in fibroblast growth factor (FGF) binding and signaling. Smooth muscle cell perlecan was decorated with both heparan sulfate and chondroitin sulfate, whereas endothelial perlecan contained exclusively heparan sulfate chains. Smooth muscle cells bound to the protein core of perlecan only when the glycosaminoglycans were removed, and this binding involved a novel site in domain III as well as domain V/endorepellin and the α2β1 integrin. In contrast, endothelial cells adhered to the protein core of perlecan in the presence of glycosaminoglycans. Smooth muscle cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains and promoted the signaling of FGF2 but not FGF1. Also endothelial cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains, but in contrast, promoted the signaling of both growth factors. Based on this differential bioactivity, we propose that perlecan synthesized by smooth muscle cells differs from that synthesized by endothelial cells by possessing different signaling capabilities, primarily, but not exclusively, due to a differential glycanation. The end result is a differential modulation of cell adhesion, proliferation and growth factor signaling in these two key cellular constituents of blood vessels.

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Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.

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