Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans.
The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with [35S]sulfate and [3H] glucosamine for 24 h and then extracted and analyzed. There was a 1.68 +/- 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of approximately 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of approximately 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 +/- 0.2-fold in media and 3.2 +/- 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.
Calvo
J C
JC
Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Rodbard
D
D
Katki
A
A
Chernick
S
S
Yanagishita
M
M
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology*; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003907:Dexamethasone / Q000494:pharmacology*; D004187:Disaccharides / Q000302:isolation & purification; D005944:Glucosamine / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D011865:Radioisotope Dilution Technique; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014316:Tritium
D011508:Chondroitin Sulfate Proteoglycans; D004187:Disaccharides; D006025:Glycosaminoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014316:Tritium; D003907:Dexamethasone; D005944:Glucosamine; D015056:1-Methyl-3-isobutylxanthine