Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone.
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.
D000818:Animals; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000378:metabolism; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006497:Heparan Sulfate / Q000378:metabolism; D008970:Molecular Weight; D011509:Proteoglycans / Q000096:biosynthesis*; D051381:Rats; D011916:Rats, Inbred F344; D013431:Sulfates / Q000378:metabolism; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology*; D013997:Time Factors
D002809:Chondroitin Sulfates; D003545:Cysteine; D006497:Heparan Sulfate; D011509:Proteoglycans; D013431:Sulfates; D013972:Thyrotropin