A Subpopulation of Fibroblast Growth Factor-2-Binding Heparan Sulfate is Lost in Human Papillary Thyroid Carcinomas

Abstract

We hypothesized that a change in composition of proteoglycans can regulate the bioactivity of fibroblast growth factor (FGF)-2 in the thyroid. In order to test this hypothesis, we established a simple and sensitive method for detecting FGF-2-binding heparan sulfates and characterized them in papillary thyroid carcinomas and normal thyroids. The thyroid extracts were applied to a Q-Sepharose anion exchange column. After the column was washed with 10 mM of phosphate buffer, 1 microgram of human recombinant FGF-2 was added onto the column. The column was eluted with a gradient of NaCl (0.3-1.5 M). Each fraction was blotted onto nitrocellulose membrane. Immunoreactivity of heparan sulfate and FGF-2 was revealed by the incubation of membranes with the specific antibodies, and quantitatively estimated by measuring the density of the color product. In normal thyroids, immunoreactivity of heparan sulfate was detected as two peaks at 0.7 and 0.9 M of NaCl. Heparan sulfate-containing fractions also showed FGF-2 immunoreactivity, indicating the complex formation of FGF-2 and heparan sulfate. In papillary thyroid carcinomas, immunoreactivity of heparan sulfate showed various elution profiles on Q-Sepharose chromatography, including single peak at 0.7 M of NaCl and the one similar to that of the normal thyroids. However, FGF-2 immunoreactivity was detected only in the fractions eluting at 0.7 M of NaCl. This loss of a subpopulation of FGF-2-binding heparan sulfate in human papillary thyroid carcinomas may lead to the increase of free FGF-2 bioavailable in extracellular matrix.

Reduced sulfation of chondroitin sulfate in thyroglobulin derived from human papillary thyroid carcinomas

“…This elution point was consistent with the peak of CS, which is mainly derived from the extracellular matrix of the thyroid. HS, which are also derived from the extracellular matrix, showed two peaks as expected; ( 10 ) the small peak at 0.9 M was coincident with the Tg and CS peaks. Treatment with heparitinase (Fig.…”

“…In the course of our earlier investigations into the regulation of the bioactivity of FGF‐2 in the thyroid, ( 17–19 ) we characterized the FGF‐2‐binding HS proteoglycans. ( 10 ) As we reported, in normal thyroids, the elution profile of FGF‐2 HS by Q‐Sepharose anion exchange column chromatography showed two peaks, one at 0.7 M and one at 0.9 M NaCl. However, most of the papillary carcinomas that we examined showed a single peak of HS at 0.7 M NaCl and none at 0.9 M NaCl.…”

“…Proteoglycans containing Tg were extracted from human thyroid tissues as described previously. ( 10 ) Briefly, thyroid tissues (1–2 g) were homogenized in 5.4 M guanidine HCl, 100 mM 6‐aminohexanoic acid and 5 mM benzamidine‐HCl using a polytron homogenizer in five times the tissue volume. The suspension was centrifuged and 1 mL of the supernatant was diluted with 9 mL of 8 M urea, 50 mM sodium acetate, 50 mM sodium ethylenediaminetetraacetic acid, 0.3 M NaCl and 0.5% Triton X‐100 at pH 6.0 (8 M urea buffer), and applied to a 0.5‐mL Q‐Sepharose anion exchange column (Pharmacia Biotech, Uppsala, Sweden) that was equilibrated with the 8 M urea buffer.…”

“…Quantitative immunoblotting was used to detect CS, HS and Tg in the above fractions as we described previously. ( 10 ) Fractions (10–100 µL) were slot‐blotted onto Hybond‐C Extra supported nitrocellulose membranes (pore size = 0.45 µm; Amersham Pharmacia Biotech, Uppsala, Sweden) using a Bio‐Dot SF microfiltration apparatus (Bio‐Rad Laboratories, Hercules, CA, USA). Each membrane was incubated separately with monoclonal anti‐Tg antibody, antichondroitin‐6‐sulfate antibody and anti‐HS antibody in TBS (10 mM Tris‐HCl, 0.15 M NaCl, pH 7.4) along with 5% skim milk at 4°C overnight.…”

Amelogenin binds to both heparan sulfate and bone morphogenetic protein 2 and pharmacologically suppresses the effect of noggin

“…4A). It has also been reported that FGF binding to heparin was abrogated by 0.5 to 0.9 M NaCl [35,36] and amelogenin binding to heparin was dissociated in 0.9 M NaCl in the present study (Fig. 4B).…”