Elke Schönherr

Proteoglycans of the extracellular matrix and growth control

Regulated cell growth results from the biological balance between soluble growth‐regulating factors, their receptors and the elicited signal cascade on the one hand side and from extracellular macromolecular components and their interplay with membrane receptors on the other side. Proteoglycans have recently been recognized not only to play a part in providing shape and biomechanical strength of organs and tissues, but also to exhibit direct and indirect cell signalling properties. In this review, we discuss the direct growth‐regulating role of proteoglycans with special emphasis on the lectican family and on the family of small proteoglycans with leucine‐rich repeats (SLRPs). Indirect actions of proteoglycans by modulation of growth factor activities and growth factor distribution are exemplified by discussing the TGF‐β‐binding properties of SLRPs and the interactions of core proteins of matrix proteoglycans with other growth factors. It is emphasized that the modulatory role of proteoglycans on cell proliferation cannot be separated from their participation in tissue organization in general, thereby explaining the diverse and sometimes contradictory reports on the effects of proteoglycans on cell proliferation and differentiation.

Decorin, a Novel Player in the Insulin-like Growth Factor System

Decorin is a multifunctional proteoglycan that is expressed by sprouting endothelial cells. Its expression supports capillary formation and cell survival. Previously, it was shown that some effects of decorin are mediated by protein kinase B and the cyclin-dependent kinase inhibitor, p21. However, the cell surface receptor responsible for these effects was unknown. We demonstrate that decorin binds to the insulin-like growth factor-I (IGF-I) receptor on endothelial cells with an affinity in the nanomolar range (KD = 18 nm), which is comparable with IGF-I (KD = 1.2 nm). Furthermore, decorin can bind IGF-I itself, but with a lower affinity (KD = 190 nm) than classical IGF-I-binding proteins. Decorin addition causes IGF-I receptor phosphorylation and activation, which is followed by receptor down-regulation. These effects are caused by the core protein of decorin, and the binding region could be mapped to the N terminus of the molecule. The physiological relevance of the decorin/IGF-I receptor interaction was corroborated in two animal models (e.g. inflammatory angiogenesis in the cornea and unilateral ureteral obstruction). In both models the IGF-I receptor was up-regulated in decorin-deficient mice compared with controls and the up-regulation could not compensate the decorin deficiency in the disease models. These data indicate that decorin is an important player in the IGF system and its loss cannot fully be compensated in different types of diseases.

Absence of Decorin Adversely Influences Tubulointerstitial Fibrosis of the Obstructed Kidney by Enhanced Apoptosis and Increased Inflammatory Reaction

Decorin, a small dermatan-sulfate proteoglycan, participates in extracellular matrix assembly and influences directly and indirectly cell behavior via interactions with signaling membrane receptors and transforming growth factor (TGF)-beta. We have therefore compared the development of tubulointerstitial kidney fibrosis in wild-type (WT) and decorin-/- mice in the model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin-/- mice did not differ in any aspect from their WT counterparts. However, already 12 hours after obstruction decorin-/- animals showed lower levels of p27(KIP1) and soon thereafter a more pronounced up-regulation and activation of initiator and effector caspases followed by enhanced apoptosis of tubular epithelial cells. Later, a higher increase of TGF-beta1 became apparent. After 7 days, there was an up to 15-fold transient up-regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan-expressing mononuclear cells. Other small proteoglycans showed no similar response. Because of enhanced degradation of type I collagen, end-stage kidneys from decorin-/- animals were more atrophic than WT kidneys. These data suggest that decorin exerts beneficial effects on tubulointerstitial fibrosis, primarily by influencing the expression of a key cyclin-dependent kinase inhibitor and by limiting the degree of apoptosis, mononuclear cell infiltration, tubular atrophy, and expression of TGF-beta1.

Selective inactivity of TGF-β/decorin complexes

Previous studies had shown that binding of TGF‐β to the small proteoglycan decorin results in its inactivation. Indeed, in osteosarcoma cells the addition of decorin prevented the TGF‐β1‐mediated up‐regulation of biglycan synthesis. However, the down‐regulation of proteoglycan‐100 remained unaltered. Even in the presence of a 100,000‐fold molar excess of decorin, TGF‐β1 was fully active in U937 monocytes with respect to the inhibition of cell proliferation. There was no inhibition of the TGF‐β‐mediated stimulation of the retraction of fibroblast‐populated collagen lattices. Thus, the formation of TGF‐β/decorin complexes leads to the neutralization of distinct effects only.

Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican, and fibromodulin

Small leucine‐rich proteoglycans (SLRPs), for example, decorin, biglycan, fibromodulin, and lumican, are extracellular matrix organizers and binding partners of TGF‐β. Decorin is also involved in growth control and angiogenesis. Hence, these proteoglycans are likely of importance in the pathogenesis of diabetic glomerulosclerosis. In normal kidney, SLRPs were preferentially expressed in the tubulointerstitium. Weak expression occurred in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, by distal tubular cells and in collecting ducts. In all stages of diabetic nephropathy, there was a marked up‐regulation of the proteoglycans in tubulointerstitium and glomeruli. Decorin and lumican became expressed in tubuli. However, in glomeruli, overexpression was not mirrored by local proteoglycan accumulation except in advanced nephropathy. In severe glomerulosclerosis, increased decorin concentrations were found in plasma and urine, and urinary TGF‐β/decorin complexes could be demonstrated indirectly. The failure to detect an increased glomerular proteoglycan quantity during the development of nephropathy could be explained by assuming that they are secreted into the mesangial matrix, but cleared via the vasculature or the urinary tract, in part as complexes with TGF‐β. They could thereby counteract the vicious circle being characterized by increased TGF‐β production and increased matrix deposition in diabetic nephropathy.

The glycosaminoglycan chain of decorin plays an important role in collagen fibril formation at the early stages of fibrillogenesis

Decorin is a multifunctional small leucine‐rich proteoglycan involved in the regulation of collagen fibrillogenesis. In patients with a variant of Ehlers–Danlos syndrome, about half of the secreted decorin lacks the single glycosaminoglycan side chain. Notably, these patients have a skin‐fragility phenotype that resembles that of decorin null mice. In this study, we investigated the role of glycanated and unglycanated decorin on collagen fibrillogenesis. Glycosaminoglycan‐free decorin, generated by mutating Ser4 of the mature protein core into Ala (DCN‐S4A), showed reduced inhibition of fibrillogenesis compared with the decorin proteoglycan. Interestingly, using a 3D matrix generated by decorin‐null fibroblasts, an increase in fibril diameter was found after the addition of decorin, and even greater effects were observed with DCN‐S4A. To avoid potential side effects of artificial tags, adenoviruses containing decorin and DCN‐S4A were used to transduce decorin‐null fibroblasts prior to matrix formation. Both molecules were efficiently incorporated into the matrix, with no changes in collagen composition and network formation, or altered expression of the related proteoglycan biglycan. Both decorin and DCN‐S4A mutants increased the collagen fibril diameter, with the latter showing the most prominent effects. These data show that at early stages of fibrillogenesis, the glycosaminoglycan chain of decorin has a reducing effect on collagen fibril diameter.

Critical Role of Glutamate in a Central Leucine-rich Repeat of Decorin for Interaction with Type I Collagen

The chondroitin/dermatan sulfate proteoglycan decorin is known to interact via its core protein with fibrillar collagens, thereby influencing the kinetics of fibril formation and the final diameter of the fibrils. To define the binding site(s) for type I collagen along the core protein, which is mainly composed of leucine-rich repeat structures, decorin cDNAs were constructed and expressed in human kidney 293 cells. The constructs encoded (i) C-terminally truncated molecules, (ii) core proteins with deletions of selected leucine-rich repeats, or (iii) various point mutations. The deletion of the sixth leucine-rich repeat Met176–Lys201 and the mutation E180K drastically interfered with the binding to reconstituted type I collagen fibrils. In contrast, the deletion of the seventh repeat Leu202–Ser222 led at the most to a marginally impaired binding, although the secretion of this proteoglycan was abnormally low. Decorin with two other point mutations in the sixth leucine-rich repeat, Lys187 → Gln and Lys200→ Gln, respectively, bound type I collagen either normally or even better than the normal recombinant proteoglycan. These data suggest that a major collagen-binding site of decorin is located within the sixth leucine-rich repeat and that glutamate-180 within this repeat is of special importance for ionic interactions between the two matrix components.

In vivo selective and distant killing of cancer cells using adenovirus-mediated decorin gene transfer

Decorin is a well‐known, ubiquitous proteoglycan that is a normal component of the ECM. Upon transgenic expression of decorin, tumor cells with diverse histogenetic background overexpress p21WAF1, a potent inhibitor of cyclin‐dependent kinase activity, become arrested in G1, and fail to generate tumors in immunocompromised animals. Because decorin is a secreted protein, it has been recently suggested that decorin could act as an autocrine and paracrine regulator of tumor growth. Here, we demonstrate that adenovirus (Ad)‐mediated transfer and expression of human decorin cDNA induced in vivo apoptosis of xenograft tumor cells in nude mice. This oncolytic activity was observed when the Ad vector encoding the decorin cDNA was injected intratumorally (i.t.) or i.v. Importantly, i.t. injection of the decorin Ad vector led to growth inhibition of the injected tumor associated with similar growth inhibition of a distant contralateral tumor, demonstrating a distant decorin antitumoral effect. Immunochemistry against human decorin and decorin quantitation in tumors confirmed that decorin migrated to the tumor distant site. Furthermore, decorin effect was specific to tumor cells, because neither apoptosis nor growth inhibition were observed in nontumoral human cells such as hepatocytes, endothelial cells, and fibroblasts, despite p21 overexpression.

Decorin deficiency leads to impaired angiogenesis in injured mouse cornea

Small leucine-rich proteoglycans play important roles in the organization of the extracellular matrix as well as for the regulation of cell behavior; two biological processes that are essential for angiogenesis. We investigated consequences of the targeted ablation of decorin (DCN), biglycan (BGN) and fibromodulin (FMOD) genes on inflammation-induced angiogenesis in the cornea. In wild-type mice, DCN was localized exclusively to the corneal stroma, while FMOD and BGN were more prominently expressed in epithelial cells. Endothelial cells from limbus blood vessels expressed BGN and FMOD, but no DCN. However, after induction of angiogenesis by chemical cauterization, DCN was expressed in the newly formed capillaries, together with BGN and FMOD. Notably, in DCN-deficient mice, the growth of vessels was significantly diminished, whereas it did not significantly change in FMOD- or BGN-deficient animals. Moreover, blood vessels of DCN-deficient mice exhibited a similar expression level of BGN as control mice, while FMOD was increased on day 3 after injury. These results indicate that DCN, in addition to its effects on fibrillogenesis, plays a regulatory role in angiogenesis and that FMOD in endothelial cells may be able to partially substitute for DCN.

Decorin Regulates Endothelial Cell Motility on Collagen I through Activation of Insulin-like Growth Factor I Receptor and Modulation of α2β1 Integrin Activity

The proteoglycan decorin is expressed by sprouting but not quiescent endothelial cells, and angiogenesis is dysregulated in its absence. Previously, we have shown that decorin core protein can bind to and activate insulin-like growth factor-I receptor (IGF-IR) in endothelial cells. In this study, we show that decorin promotes α2β1 integrin-dependent endothelial cell adhesion and migration on fibrillar collagen type I. We provide evidence that decorin modulates cell-matrix interaction in this context by stimulating cytoskeletal and focal adhesion reorganization through activation of the IGF-IR and the small GTPase Rac. Further, the glycosaminoglycan moiety of decorin interacts with α2β1, but not α1β1 integrin, at a site distinct from the collagen I-binding A-domain, to allosterically modulate collagen I-binding activity of the integrin. We propose that induction of decorin expression in angiogenic, as opposed to quiescent, endothelial cells promotes a motile phenotype in an interstitial collagen I-rich environment by both signaling through IGF-IR and influencing α2β1 integrin activity.