Pharmacokinetics and disposition characteristics of recombinant decorin after intravenous injection into mice.
The pharmacokinetics and disposition characteristics of recombinant decorin after intravenous administration were investigated in mice. Following bolus injection of 111In-labeled decorin at doses of 0.02 and 0.1 mg/kg, radioactivity rapidly disappeared from the circulation and approximately 70% of the dose accumulated in liver within 10 min. 111In-labeled decorin was preferentially localized in hepatic nonparenchymal cells. At a higher dose of 1 mg/kg, clearance from the circulation and hepatic uptake of [111In]decorin were slower than at lower doses. Both the accumulation in other tissues and urinary excretion of [111In]decorin were 5% or less. Pharmacokinetic analysis demonstrated that hepatic uptake clearance was large and accounted almost completely for total body clearance; in addition the clearance values decreased as the dose increased, suggesting that the hepatic uptake of decorin is mediated by a specific mechanism which becomes saturated at higher doses. In competitive inhibition experiments, hepatic uptake of 111In-labeled decorin was partially inhibited (about 20-30%) by several sulfated glycans such as glycosaminoglycans and dextran sulfate and by mannosylated bovine serum albumin (BSA), mannan and mannose to a lesser extent (about 10%). On the other hand, polyinosinic acid, polycytidylic acid and succinylated BSA were ineffective, suggesting that the scavenger receptor for polyanions in the liver is not involved in the hepatic uptake of decorin. A basic protein, protamine, and a ligand of the apoE receptor, lactoferrin, also had no effect. Taken together, the present results have demonstrated that recombinant decorin is rapidly eliminated from the blood circulation through extensive uptake by the liver, primarily by the nonparenchymal cells, following systemic administration. The sugar structure and mannose residue in decorin have also been suggested to play an important role in the hepatic uptake of decorin. These findings provide useful information for the development of decorin as a therapeutic agent. (+info)
Cooperative action of germ-line mutations in decorin and p53 accelerates lymphoma tumorigenesis.
Ectopic expression of decorin in a wide variety of transformed cells results in growth arrest and the inability to generate tumors in nude mice. This process is caused by a decorin-mediated activation of the epidermal growth factor receptor, which leads to a sustained induction of endogenous p21(WAF1/CIP1) (the cyclin-dependent kinase inhibitor p21) and growth arrest. However, mice harboring a targeted disruption of the decorin gene do not develop spontaneous tumors. To test the role of decorin in tumorigenesis, we generated mice lacking both decorin and p53, an established tumor-suppressor gene. Mice lacking both genes showed a faster rate of tumor development and succumbed almost uniformly to thymic lymphomas within 6 months [mean survival age (T50) approximately 4 months]. Mice harboring one decorin allele and no p53 gene developed the same spectrum of tumors as the double knockout animals, but had a survival rate similar to the p53 null animals (T50 approximately 6 months). Ectopic expression of decorin in thymic lymphoma cells isolated from double mutant animals markedly suppressed their colony-forming ability. When these lymphoma cells were cocultured with fibroblasts derived from either wild-type or decorin null embryos, the cells grew faster in the absence of decorin. Moreover, exogenous decorin proteoglycan or its protein core significantly retarded their growth in vitro. These results indicate that the lack of decorin is permissive for lymphoma tumorigenesis in a mouse model predisposed to cancer and suggest that germ-line mutations in decorin and p53 may cooperate in the transformation of lymphocytes and ultimately lead to a more aggressive phenotype by shortening the tumor latency. (+info)
Fatty acids modulate the composition of extracellular matrix in cultured human arterial smooth muscle cells by altering the expression of genes for proteoglycan core proteins.
In diabetes-associated microangiopathies and atherosclerosis, there are alterations of the extracellular matrix (ECM) in the intima of small and large arteries. High levels of circulating nonesterified fatty acids (NEFAs) are present in insulin resistance and type 2 diabetes. High concentrations of NEFAs might alter the basement membrane composition of endothelial cells. In arteries, smooth muscle cells (SMCs) are the major producers of proteoglycans and glycoproteins in the intima, and this is the site of lipoprotein deposition and modification, key events in atherogenesis. We found that exposure of human arterial SMCs to 100-300 micromol/albumin-bound linoleic acid lowered their proliferation rate and altered cell morphology. SMCs expressed 2-10 times more mRNA for the core proteins of the proteoglycans versican, decorin, and syndecan 4 compared with control cells. There was no change in expression of fibronectin and perlecan. The decorin glycosaminoglycan chains increased in size after exposure to linoleic acid. The ECM produced by cells grown in the presence of linoleic acid bound 125I-labeled LDL more tightly than that of control cells. Darglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, neutralized the NEFA-mediated induction of the decorin gene. This suggests that some of the NEFA effects are mediated by PPAR-gamma. These actions of NEFAs, if present in vivo, could contribute to changes of the matrix of the arterial intima associated with micro- and macroangiopathies. (+info)
Connective tissues: matrix composition and its relevance to physical therapy.
In the last 2 decades, the understanding of CT structure and function has increased enormously. It is now clear that the cells of the various CTs synthesize a variety of ECM components that act not only to underpin the specific biomechanical and functional properties of tissues, but also to regulate a variety of cellular functions. Importantly for the physical therapist, and as discussed above, CTs are responsive to changes in the mechanical environment, both naturally occurring and applied. The relative proportions of collagens and PGs largely determine the mechanical properties of CTs. The relationship between the fibril-forming collagens and PG concentration is reciprocal. Connective tissues designed to resist high tensile forces are high in collagen and low in total PG content (mostly dermatan sulphate PGs), whereas CTs subjected to compressive forces have a greater PG content (mostly chondroitin sulphate PGs). Hyaluronan has multiple roles and not only provides tissue hydration and facilitation of gliding and sliding movements but also forms an integral component of large PG aggregates in pressure-resisting tissues. The smaller glycoproteins help to stabilize and link collagens and PGs to the cell surface. The result is a complex interacting network of matrix molecules, which determines both the mechanical properties and the metabolic responses of tissues. Patients with CT problems affecting movement are frequently examined and treated by physical therapists. A knowledge of the CT matrix composition and its relationship to the biomechanical properties of these tissues, particularly the predictable responses to changing mechanical forces, offers an opportunity to provide a rational basis for treatments. The complexity of the interplay among the components, however, requires that further research be undertaken to determine more precisely the effects of treatments on the structure and function of CTs. (+info)
Initiation of galactosaminoglycan biosynthesis. Separate galactosylation and dephosphorylation pathways for phosphoxylosylated decorin protein and exogenous xyloside.
By using various radiolabelled precursors, glycosylation and phosphorylation of decorin in a rat fibroblast cell line was investigated in the presence of increasing concentrations of p-nitrophenyl-O-beta-d-xylopyranoside. Decorin core protein glycanation was suppressed to approximately 25% of the normal level in the presence of 2 mm and 3 mm xyloside. Glycans/saccharides were released from the core protein and size-separated by gel chromatography. The intracellular decorin obtained from cells treated with 2 mm xyloside was substituted with Xyl and also with Gal-Xyl and Gal-Gal-Xyl, but not with longer saccharides. Only the trisaccharide contained an almost fully phosphorylated Xyl. We conclude that galactosylation of endogenous, xylosylated decorin and exogenous xyloside probably follow separate pathways or that xylosides and early decorin glycoforms are kept separated. At the addition of the first glucuronic acid the two pathways seem to merge and dephosphorylation of decorin takes place. Xyloside-primed and secreted galactosaminoglycan chains produced simultanously retained phosphorylated Xyl. Inadequate dephosphorylation could be due to excess substrate or to a short transit.time. As shown previously [Moses, J., Oldberg, A., Eklund, E. & Fransson, L.-A. (1997) Eur. J. Biochem. 248, 767-774], brefeldin A-arrested decorin is substituted with the linkage-region extended with an undersulphated and incomplete galactosaminoglycan chain. In cells treated with this drug, xylosides were unable to prime galactosaminoglycan synthesis and unable to inhibit glycosylation and phosporylation of decorin. (+info)
Distinct secondary structures of the leucine-rich repeat proteoglycans decorin and biglycan. Glycosylation-dependent conformational stability.
Biglycan and decorin have been overexpressed in eukaryotic cells and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans (Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the final form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that in this specific domain the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provide further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1 and 2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of stable unfolding intermediates. (+info)
Decorin is a Zn2+ metalloprotein.
Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by leucine-rich repeat motifs. We show here that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells has a high affinity for Zn2+ as demonstrated by equilibrium dialyses. The Zn2+-binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in a spacing reminiscent of a zinc finger. A recombinant 41-amino acid long peptide representing the N-terminal domain of decorin has full Zn2+ binding activity and binds two Zn2+ ions with an average KD of 3 x 10(-7) M. Binding of Zn2+ to this peptide results in a change in secondary structure as shown by circular dichroism spectroscopy. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn2+-binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, do not bind Zn2+ with high affinity. (+info)
Resistance of small leucine-rich repeat proteoglycans to proteolytic degradation during interleukin-1-stimulated cartilage catabolism.
A bovine nasal-cartilage culture system has been utilized to analyse the catabolic events occurring in response to interleukin-1beta over a 14-day period. An early event following the start of interleukin-1 treatment was the release of glycosaminoglycan into the culture medium. This release was accompanied by the appearance in the tissue, and shortly thereafter also in the culture media, of a globular domain (G1)-containing aggrecan degradation product generated by the action of aggrecanase. Link protein was also released from the cartilage with a similar timeframe to that of the G1 fragment, although there was no evidence of its proteolytic degradation. By comparison with aggrecan, the small leucine-rich repeat proteoglycans decorin, biglycan and lumican showed a resistance to both proteolytic cleavage and release throughout the culture period. In contrast, fibromodulin exhibited a marked decrease in size after day 4, presumably due to proteolytic modification, but the major degradation product was retained throughout the culture period. Also in contrast with the early changes in the components of the proteoglycan aggregate, type II collagen did not display signs of extensive degradation until much later in the culture period. Collagen degradation products compatible with collagenase action first appeared in the medium by day 10 and increased thereafter. These data demonstrate that the leucine-rich repeat proteoglycans are resistant to proteolytic action during interleukin-1-stimulated cartilage catabolism, compared with aggrecan. This resistance and continued interaction with the surface of the collagen fibrils may help to stabilize the collagen fibrillar network and protect it from extensive proteolytic attack during the early phases of cartilage degeneration. (+info)