Abstract
A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.
Highlights
From the Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892 and the *Division of Endocrinology, Toranomon Hospital, and Okinakn Memorial Institute for Medical Research, Tokyo 105, Japan
Single chain glycosaminoglycans releasedby papaindigestion or alkaline borohydride treatmentof either theCS or HS proteoglycans had average molecular weights of approximateIy 30,000 on Sepharose CL-GB chromatography. Both chondroitin sulfate (CS) and HS proteoglycans were relatively small and containedonly one or two glycosaminoglycans chains. 3H and 35Sincorporation into both CS and HS proteoglycans were increased by thyroidstimulating hormone (TSH)in a dose-dependent manner, whichis in part explainebdy an adenylatecyclasedependent mechanism as indicated by a similar effect
Proteoglycans may be involved in abnormal cell growth in thyroid tissue, little is known about proteoglycans in thyroidcells in culture
Summary
5, pp. 1745-1754, 1988 Printed in U.S.A. Characterization of Proteoglycans Synthesizedby Rat ThyroidCells in Culture and Their Responseto Thyroid-stimulating Hormone*. ’ The abbreviations used are: TSH, thyroid-stimulating hormone; FRTL, a Fisher rat thyroid cell line maintained in low serum; FCS, fetal calf serum; ADi-4S, 2-acetamido-2-deoxy-3-O-[/3-~-gluco-4-enepyranosyluronic acid]-4-O-sulfo-D-galactoseA;Di-6S, 2-acetamido-2deoxy-3-O-[~-~-gluco-4-enepyranosyluroanciicdl-6-O-sulfo-~-galactose; CS, chondroitin sulfate; HS, heparan sulfate; Bt2cAMP, dibutyryl cyclic AMP; HPLC, high performance liquid chromatography. The reaction was stopped by neutralization with glacial acetic acid on ice. Alkaline borohydride-treated samples were applied to a column of Sepharose CL-GB eluted with 4 M guanidine HCl buffer containing 0.5% Triton X-100. The amounts of radiolabeled proteoglycans in each medium and cell layer compartment were determined as described above
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