Rick T. Owens

Ace Is a Collagen-binding MSCRAMM from Enterococcus faecalis

A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM fromStaphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174–319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151–318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174–319) suggested a structure very similar to that reported for residues 151–318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a β-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 10, 833–838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.

Decorin is a novel antagonistic ligand of the Met receptor

Decorin, a member of the small leucine-rich proteoglycan gene family, impedes tumor cell growth by down-regulating the epidermal growth factor receptor. Decorin has a complex binding repertoire, thus, we predicted that decorin would modulate the bioactivity of other tyrosine kinase receptors. We discovered that decorin binds directly and with high affinity (Kd = ∼1.5 nM) to Met, the receptor for hepatocyte growth factor (HGF). Binding of decorin to Met is efficiently displaced by HGF and less efficiently by internalin B, a bacterial Met ligand. Interaction of decorin with Met induces transient receptor activation, recruitment of the E3 ubiquitin ligase c-Cbl, and rapid intracellular degradation of Met (half-life = ∼6 min). Decorin suppresses intracellular levels of β-catenin, a known downstream Met effector, and inhibits Met-mediated cell migration and growth. Thus, by antagonistically targeting multiple tyrosine kinase receptors, decorin contributes to reduction in primary tumor growth and metastastic spreading.

Decorin Protein Core Inhibits in Vivo Cancer Growth and Metabolism by Hindering Epidermal Growth Factor Receptor Function and Triggering Apoptosis via Caspase-3 Activation

Decorin is not only a regulator of matrix assembly but also a key signaling molecule that modulates the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR). Decorin evokes protracted internalization of the EGFR via a caveolar-mediated endocytosis, which leads to EGFR degradation and attenuation of its signaling pathway. In this study, we tested if systemic delivery of decorin protein core would affect the biology of an orthotopic squamous carcinoma xenograft. After tumor engraftment, the animals were given intraperitoneal injections of either vehicle or decorin protein core (2.5-10 mg kg-1) every 2 days for 18-38 days. This regimen caused a significant and dose-dependent inhibition of the tumor xenograft growth, with a concurrent decrease in mitotic index and a significant increase in apoptosis. Positron emission tomography showed that the metabolic activity of the tumor xenografts was significantly reduced by decorin treatment. Decorin protein core specifically targeted the tumor cells enriched in EGFR and caused a significant down-regulation of EGFR and attenuation of its activity. In vitro studies showed that the uptake of decorin by the A431 cells was rapid and caused a protracted down-regulation of the EGFR to levels similar to those observed in the tumor xenografts. Furthermore, decorin induced apoptosis via activation of caspase-3. This could represent an additional mechanism whereby decorin might influence cell growth and survival.

Genetic Evidence for the Coordinated Regulation of Collagen Fibrillogenesis in the Cornea by Decorin and Biglycan

Decorin and biglycan are class I small leucine-rich proteoglycans (SLRPs) involved in regulation of collagen fibril and matrix assembly. We hypothesize that tissue-specific matrix assembly, such as in the cornea, requires a coordinate regulation involving multiple SLRPs. To this end, we investigated the expression of decorin and biglycan in the cornea of mice deficient in either SLRP gene and in double-mutant mice. Decorin and biglycan exhibited overlapping spatial expression patterns throughout the corneal stroma with differential temporal expression. Whereas decorin was expressed at relatively high levels in all developmental stages, biglycan expression was high early, decreased during development, and was present at very low levels in the mature cornea. Ultrastructural analyses demonstrated comparable fibril structure in the decorin- and biglycan-null corneas compared with wild-type controls. We found a compensatory up-regulation of biglycan gene expression in the decorin-deficient mice, but not the reverse. Notably, the corneas of compound decorin/biglycan-null mice showed severe disruption in fibril structure and organization, especially affecting the posterior corneal regions, corroborating the idea that biglycan compensates for the loss of decorin. Fibrillogenesis assays using recombinant decorin and biglycan confirmed a functional compensation, with both having similar effects at high SLRP/collagen ratios. However, at low ratios decorin was a more efficient regulator. The use of proteoglycan or protein core yielded comparable results. These findings provide firm genetic evidence for an interaction of decorin and biglycan during corneal development and further suggest that decorin has a primary role in regulating fibril assembly, a function that can be fine-tuned by biglycan during early development.

A Porcine-Derived Acellular Dermal Scaffold That Supports Soft Tissue Regeneration: Removal of Terminal Galactose-α-(1,3)-Galactose and Retention of Matrix Structure

Sub-optimal clinical outcomes after implantation of animal-derived tissue matrices may be attributed to the nature of the processing of the material or to an immune response elicited in response to xenogeneic epitopes. The ability to produce a porcine-derived graft that retains the structural integrity of the extracellular matrix and minimizes potential antigenic response to galactose-alpha-(1,3)-galactose terminal disaccharide (alpha-Gal) may allow the scaffold to support regeneration of native tissue. Dermal tissue from wild-type (WT-porcine-derived acellular dermal matrix [PADM]) or Gal-deficient (Gal(-/-) PADM) pigs was processed to remove cells and DNA while preserving the structural integrity of the extracellular matrix. In addition, the WT tissue was subjected to an enzymatic treatment to minimize the presence of alpha-Gal (Gal-reduced PADM). Extracellular matrix composition and integrity was assessed by histological, immunohistochemical (IHC), and ultrastructural analysis. In vivo performance was evaluated by implantation into the abdominal wall of Old World primates in an exisional repair model. Anti-alpha-Gal activity in the serum of monkeys implanted subcutaneously was assessed by ELISA. Minimal modification to the extracellular matrix was assessed by evaluation of intact structure as demonstrated by staining patterns for type I and type VII collagens, laminin, and fibronectin similar to native porcine skin tissues. Explants from the abdominal wall showed evidence of remodeling, notably fibroblast cell repopulation and revascularization, as early as 1 month. Serum ELISA revealed an initial anti-alpha-Gal induction that decreased to baseline levels over time in the primates implanted with WT-PADM, whereas no or minimal anti-Gal activity was detected in the primates implanted with Gal(-/-) PADM or Gal-reduced PADM. The combination of a nondamaging process, successful removal of cells, and reduction of xenogeneic alpha-Gal antigens from the porcine dermal matrix are critical for producing a material with the ability to remodel and integrate into host tissue and ultimately support soft tissue regeneration.

An Antimetastatic Role for Decorin in Breast Cancer

Decorin, a member of the small leucine-rich proteoglycan gene family, down-regulates members of the ErbB receptor tyrosine kinase family and attenuates their signaling, leading to growth inhibition. We investigated the effects of decorin on the growth of ErbB2-overexpressing mammary carcinoma cells in comparison with AG879, an established ErbB2 kinase inhibitor. Cell proliferation and anchorage-independent growth assays showed that decorin was a potent inhibitor of breast cancer cell growth and a pro-apoptotic agent. When decorin and AG879 were used in combination, the inhibitory effect was synergistic in proliferation assays but only additive in both colony formation and apoptosis assays. Active recombinant human decorin protein core, AG879, or a combination of both was administered systemically to mice bearing orthotopic mammary carcinoma xenografts. Primary tumor growth and metabolism were reduced by approximately 50% by both decorin and AG879. However, no synergism was observed in vivo. Decorin specifically targeted the tumor cells and caused a significant reduction of ErbB2 levels in the tumor xenografts. Most importantly, systemic delivery of decorin prevented metastatic spreading to the lungs, as detected by novel species-specific DNA detection and quantitative assays. In contrast, AG879 failed to have any effect. Our data support a role for decorin as a powerful and effective therapeutic agent against breast cancer due to its inhibition of both primary tumor growth and metastatic spreading.

Interactions of the Extracellular Matrix Proteoglycans Decorin and Biglycan with C1q and Collectins

Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.

Decorin Antagonizes the Angiogenic Network: CONCURRENT INHIBITION OF MET, HYPOXIA INDUCIBLE FACTOR 1α, VASCULAR ENDOTHELIAL GROWTH FACTOR A, AND INDUCTION OF THROMBOSPONDIN-1 AND TIMP3*

Background: Decorin antagonizes multiple receptor tyrosine kinases, such as Met, to suppress tumorigenesis. Results: Decorin promotes angiostasis by blocking hypoxia inducible factor-1α and β-catenin to inhibit vascular endothelial growth factor A and matrix metalloprotease-2/9 activity concurrent with thrombospondin-1 and TIMP3 induction. Conclusion: Decorin abrogates the pro-angiogenic HGF/Met signaling axis, thereby repressing vascular endothelial growth factor A-mediated angiogenesis under normoxia. Significance: Soluble decorin attenuates early tumor growth by preventing normoxic angiogenic signaling through the Met receptor. Decorin, a small leucine-rich proteoglycan, inhibits tumor growth by antagonizing multiple receptor tyrosine kinases including EGFR and Met. Here, we investigated decorin during normoxic angiogenic signaling. An angiogenic PCR array revealed a profound decorin-evoked transcriptional inhibition of pro-angiogenic genes, such as HIF1A. Decorin evoked a reduction of hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor A (VEGFA) in MDA-231 breast carcinoma cells expressing constitutively-active HIF-1α. Suppression of Met with decorin or siRNA evoked a similar reduction of VEGFA by attenuating downstream β-catenin signaling. These data establish a noncanonical role for β-catenin in regulating VEGFA expression. We found that exogenous decorin induced expression of thrombospondin-1 and TIMP3, two powerful angiostatic agents. In contrast, decorin suppressed both the expression and enzymatic activity of matrix metalloprotease (MMP)-9 and MMP-2, two pro-angiogenic proteases. Our data establish a novel duality for decorin as a suppressor of tumor angiogenesis under normoxia by simultaneously down-regulating potent pro-angiogenic factors and inducing endogenous anti-angiogenic agents.

Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, α1β1 integrin and Staphylococcus aureus Cna MSCRAMM

Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin α2β1 and a Staphylococcus aureus adhesin reveal a “trench” on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512–28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833–838). We report here the crystal structure of the α subunit I domain from the α1β1 integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human α1 integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs.

Decorin causes autophagy in endothelial cells via Peg3.

Significance We identified a function for a member of the extracellular matrix in the regulation of autophagy. Decorin, a member of the small leucine-rich proteoglycan family and an established pan-receptor tyrosine kinase inhibitor, evokes endothelial cell autophagy and inhibits angiogenesis. This process is mediated by a high-affinity interaction with VEGFR2 which leads to increased levels of Peg3, a tumor-suppressor gene. We provide mechanistic evidence that Peg3 is required to maintain basal levels of Beclin 1, a major autophagic marker. These data provide a paradigmatic shift for other soluble matrix constituents to regulate autophagy. Soluble decorin affects the biology of several receptor tyrosine kinases by triggering receptor internalization and degradation. We found that decorin induced paternally expressed gene 3 (Peg3), an imprinted tumor suppressor gene, and that Peg3 relocated into autophagosomes labeled by Beclin 1 and microtubule-associated light chain 3. Decorin evoked Peg3-dependent autophagy in both microvascular and macrovascular endothelial cells leading to suppression of angiogenesis. Peg3 coimmunoprecipitated with Beclin 1 and LC3 and was required for maintaining basal levels of Beclin 1. Decorin, via Peg3, induced transcription of Beclin 1 and microtubule-associated protein 1 light chain 3 alpha genes, thereby leading to a protracted autophagic program. Mechanistically, decorin interacted with VEGF receptor 2 (VEGFR2) in a region overlapping with its natural ligand VEGFA, and VEGFR2 was required for decorin-evoked Beclin 1 and microtubule-associated protein 1 light chain 3 alpha expression as well as for Peg3 induction in endothelial cells. Moreover, decorin induced VEGFR2-dependent mitochondrial fragmentation and loss of mitochondrial membrane potential. Thus, we have unveiled a mechanism for a secreted proteoglycan in inducing Peg3, a master regulator of macroautophagy in endothelial cells.