Synthesis Of Large Proteoglycans Research Articles

Stimulation of Small Proteoglycan Synthesis by the Hyaluronan Synthesis Inhibitor 4-Methylumbelliferone in Human Skin Fibroblasts

Human skin fibroblasts cultured with 4-methylumbelliferone (MU), a hyaluronan synthesis inhibitor, produce a hyaluronan-deficient extracellular matrix (See []). Our present study investigated the effects of MU on proteoglycan, which is the other main component of the extracellular matrix, and interacts with hyaluronan. Proteoglycans isolated from culture medium in the presence or absence of MU were characterized by gel-filtration chromatography, ion-exchange HPLC, electrophoresis, and immunoblotting. We found that MU had only a negligible effect on the synthesis of large proteoglycan but increased the production of small proteoglycan in comparison with cultures lacking MU. This small proteoglycan was identified by immunoblotting as decorin. The structures of decorin synthesized in the presence and absence of MU were compared by gel-filtration chromatography, and the data indicated that cells incubated with MU produced a larger decorin molecule than cells incubated without MU. Furthermore, the two decorins had galactosaminoglycan chains of different sizes. These results suggest that MU inhibits the synthesis of hyaluronan and accelerates production of the larger decorin in the extracellular matrix.

Effect of material geometry on cartilagenous tissue formation in vitro.

The effect of material geometry, as defined by average pore size, on chondrocyte phenotype and cartilagenous tissue formation in vitro was examined. Bovine articular chondrocytes were plated on porous titanium alloy (Ti6Al4V) discs of different average pore sizes (13, 43, and 68 microm) and grown in culture for 4 weeks. Chondrocyte phenotype was maintained as indicated by the synthesis of large proteoglycans (Kav +/- SD: 13 microm = 0.28 +/- 0.01; 43 microm = 0.29 +/- 0.01; 68 microm = 0.27 +/- 0.02) and type II collagen. Light microscopical examination of histological sections of the composites showed that cartilagenous tissue had formed on all discs. The cartilagenous tissue on the discs of the smallest average pore size (13 microm) was significantly thicker than the tissue on the discs of larger average pore sizes and also had greater amounts of proteoglycan [mean glycosaminoglycan content +/- SD microg/disc): 13 microm = 246.9 +/- 7.8; 43 microm = 190.4 +/- 10.2; 68 microm = 156.6 +/- 25.8, p = 0.002] and DNA [mean DNA content +/- SD microg/disc): 13 microm = 12.5 +/- 0.6; 43 microm = 8.3 +/- 0.2; 68 microm = 9.3 +/- 0.9, p = 0.0008]. However, the amount of proteoglycan accumulated per cell was similar in the tissues generated on the discs of different average pore sizes. In contrast, the amount of collagen in the cartilagenous tissues showed no significant differences between the different pore sizes, but the amount of collagen accumulated per cell was less in the tissue formed on the smallest pore size disc (13 microm) as compared with the tissue formed on the discs of the larger pore sizes [mean hydroxyproline content/DNA (microg/microg) +/- SD: 13 microm = 1.56 +/- 0.2; 43 microm = 2.19 +/- 0.2; 68 microm = 2.3 +/- 0.3]. These results suggest that material geometry, as defined by pore size, can affect the amount and composition of the cartilagenous tissue that forms.

The effect of compressive loading on proteoglycan turnover in cultured fetal tendon.

A fibrocartilaginous tissue develops in tendon at the point where the tendon wraps under bone and is subjected to transverse compressive loading in addition to tension. This tissue is characterized by a high level of large proteoglycan (aggrecan), which could accumulate because of increased synthesis, diminished turnover, or both. To examine the effect of loading on proteoglycan turnover segments of fetal tendon in sterile culture were subjected to cyclic, uniaxial compression loading to 30% of initial thickness once every 6 sec. for 72 h, and then allowed to incorporate 35S-sulfate for 12 h. The rate of loss of newly-synthesized 35S-proteoglycans from tissue was determined during subsequent culture for up to 12 days, with or without continued loading. Proteoglycan was lost from fetal tendon segments rapidly during the first 3 days of culture and slowly thereafter. Loss of newly-synthesized proteoglycan from adult tendon fibrocartilage was linear, with a half life of 12 d. Segments of fetal tendon subjected to cyclic compression before labeling synthesized more proteoglycan. These segments lost a greater percent of labeled proteoglycan to medium during a subsequent 12-day culture period than matched segments that had not experienced loading. Analysis of medium and tissue proteoglycans by SDS polyacrylamide gel electrophoresis and sieve chromatography indicated that small proteoglycans (decorin and biglycan) were retained in both loaded and non-loaded tissue whereas large proteoglycans (migrating in the Vo of a Sepharose CL-4B column) were readily lost. It is concluded that the 3-day loading regimen did not diminish turnover of large proteoglycan. To the contrary, although synthesis of large proteoglycan was enhanced by the loading regimen, these proteoglycans were still rapidly lost from the fetal tissue.

Steroids and IGF-1 maintain synthesis of large proteoglycans in murine articular cartilage

Steroids and IGF-1 maintain synthesis of large proteoglycans in murine articular cartilage