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Title: Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans

Abstract

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 {plus minus} 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 {plus minus} 0.2-fold in media and 3.2 {plus minus}more » 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.« less

Authors:
; ; ; ;  [1]
  1. (Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (USA))
Publication Date:
OSTI Identifier:
5510089
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; (United States); Journal Volume: 266:17
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; FAT CELLS; CELL DIFFERENTIATION; GLYCOPROTEINS; CHEMICAL COMPOSITION; ADIPOSE TISSUE; DEXAMETHASONE; DISACCHARIDES; GLUCOSAMINE; ION EXCHANGE CHROMATOGRAPHY; LIQUID COLUMN CHROMATOGRAPHY; MICE; MOLECULAR WEIGHT; SULFATES; SULFUR 35; TRITIUM; ADRENAL HORMONES; AMINES; ANIMAL CELLS; ANIMAL TISSUES; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BODY; CARBOHYDRATES; CHROMATOGRAPHY; CONNECTIVE TISSUE; CONNECTIVE TISSUE CELLS; CORTICOSTEROIDS; DAYS LIVING RADIOISOTOPES; EVEN-ODD NUCLEI; GLUCOCORTICOIDS; HEXOSAMINES; HEXOSES; HORMONES; HYDROGEN ISOTOPES; HYDROXY COMPOUNDS; ISOTOPES; KETONES; LIGHT NUCLEI; MAMMALS; MONOSACCHARIDES; NUCLEI; ODD-EVEN NUCLEI; OLIGOSACCHARIDES; ORGANIC COMPOUNDS; OXYGEN COMPOUNDS; PREGNANES; PROTEINS; RADIOISOTOPES; RODENTS; SACCHARIDES; SEPARATION PROCESSES; SOMATIC CELLS; STEROIDS; SULFUR COMPOUNDS; SULFUR ISOTOPES; TISSUES; VERTEBRATES; YEARS LIVING RADIOISOTOPES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. United States: N. p., 1991. Web.
Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., & Yanagishita, M. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. United States.
Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M. 1991. "Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans". United States. doi:.
@article{osti_5510089,
title = {Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans},
author = {Calvo, J.C. and Rodbard, D. and Katki, A. and Chernick, S. and Yanagishita, M.},
abstractNote = {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 {plus minus} 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 {plus minus} 0.2-fold in media and 3.2 {plus minus} 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.},
doi = {},
journal = {Journal of Biological Chemistry; (United States)},
number = ,
volume = 266:17,
place = {United States},
year = 1991,
month = 6
}
  • The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparinmore » and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.« less
  • Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of (/sup 35/S) sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the /sup 35/S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The isolated proteoglycans were of approx. 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. The presence in normal mammalian cells of chondroitinmore » sulfate proteoglycans that contain a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched populations of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leumekia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans are all highly sulfated, protease-resistant proteoglycans.« less
  • Monolayer cultures of embryonic chick chondrocytes were incubated with /sup 35/SO/sub 4//sup 2 -/ in the presence and absence of 1.0 mM p-nitrophenyl-..beta..-D-xyloside for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free polysaccharide chains were measured following gel filtration on Sephadex G-200. Synthesis of ..beta..-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. When levels of cartilage-specific core protein were determined by a radioimmunoassay, similar amounts of core protein were found in both ..beta..-xyloside and control cultures, indicating that decreased synthesis of core protein is not responsible formore » the observed decrease in chondroitin sulfate proteoglycan production. Activity levels of the chain-initiating glycosyltransferases (UDP-D-xylose:core protein xylosyltransferase and UDP-D-galactose:D-xylose galactosyltransferase) as well as the extent of xylosylation of core protein were found to be similar in cell extracts from both culture types. Furthermore, ..beta..-xylosides did not inhibit the xylosyltransferase reaction in cell-free studies. In contrast, the ..beta..-xylosides effectively competed with several galactose acceptors, including an enzymatically synthesized xylosylated core protein acceptor, in the first galactosyltransferase reaction.« less
  • Intracellular proteoglycans (PG), putative markers of mast cell subtype, were isolated and characterized from three dog mastocytome cell lines. Mastocytoma cells were adapted to serial passage as subcutaneous tumors in BALB/c nude mice for 10 generations. Cells were disaggregated and labeled with (number/sup 5/S)SO/sub 4/ in culture. Intracellular /sup 35/S-labeled PG were isolated by sonication in 4M guanidine containing protease and glycosidase inhibitors, then chromatographed on Sepharose CL-4B. All 3 cell lines yielded a major /sup 35/S component at the Vo (Mr greater than or equal to 10/sup 6/). Excluded fractions were treated with alkaline-borohydride and the liberated glycosaminoglycans (GAG)more » were analyzed on Sephacryl S-200. GAG chains had an apparent Mr of 40-50,000. GAG chains were subjected to chondroitinase ABC and nitrous acid degradation and the products were analyzed on Sepharcyl S-200. In one cell line (BR), 75% of /sup 35/S-labeled GAG was resistant to chondroitinase treatment, but was degraded by nitrous acid, characteristic of heparin. The remaining 25% of /sup 35/S-labeled GAG was degraded to oligosaccharides by chondroitinase indicating chondroitin sulfate (CS). A second cell line (G) also contained both heparin and CS-PG. The remaining cell line (C2) contained 2% heparin-PG and 98% CS-PG. The results demonstrate the simultaneous presence of both heparin and CS-PG in mast cells of clonal origin, and raise important questions on the role of PGs in mast cell granule storage and release mechanisms.« less
  • No abstract prepared.