Insulin-like growth factors I and II are autocrine factors in stimulating proteoglycan synthesis, a marker of differentiated chondrocytes, acting through their respective receptors on a clonal human chondrosarcoma-derived chondrocyte cell line, HCS-2/8.
Both insulin-like growth factor (IGF)-I and IGF-II increased the synthesis of cartilage-type, large proteoglycan in a human chondrosarcoma-derived chondrocyte cell line, HCS-2/8. In contrast to the stimulatory effects of IGFs on costal chondrocytes of the young rabbit, the stimulatory effect of IGF-II on proteoglycan synthesis in HCS-2/8 cells was more potent than that of IGF-I. IGF-II, but not IGF-I, increased calcium influx into HCS-2/8 cells, and there was a close relation between the stimulation of proteoglycan synthesis and the calcium influx. [125I]IGF-I bound to HCS-2/8 cells, and this binding was competitively inhibited by low concentrations of unlabeled IGF-I, higher concentrations of IGF-II, and much higher concentrations of insulin. [125I]IGF-II also bound to the cells, and its binding was competitively inhibited by IGF-II and slightly inhibited by higher concentrations of IGF-I and much higher concentrations of insulin. When radioligand-receptor complexes were separated by SDS-PAGE and subjected to autoradiography, two major bands at 260 and 130 kDa were observed, which correspond to the IGF type II receptor (IGF-IIR) and the alpha subunit of the IGF type I receptor (IGF-IR), indicating the presence of both receptors. When confluent cultures of HCS-2/8 cells were maintained in serum-free medium, proteoglycan synthesis did not decrease unless the medium was repeatedly replaced. Conditioned medium of HCS-2/8 cells stimulated the HCS-2/8 cells to synthesize proteoglycans. RIA revealed that the cells produced both IGF-II and IGF-I. Transcripts of messenger RNAs of both IGF-I and IGF-II and both IGF-IR and IGF-IIR also were detectable by Northern analysis. Both anti-IGF-IR antibody and anti-IGF-II antibody inhibited proteoglycan synthesis. Mannose-6-phosphate, which is known to bind to IGF-IIR, stimulated proteoglycan synthesis, potentiated IGF-II-stimulated proteoglycan synthesis, and enhanced the binding affinity for IGF-II but not for IGF-I. Even in the presence of anti-IGF-IR antibody, IGF-II and mannose-6-phosphate stimulated proteoglycan synthesis in the cells. [Leu27]IGF-II, an IGF-II analogue with high affinity only for IGF-IIR, strongly stimulated proteoglycan synthesis in HCS-2/8 cells but [Arg54, Arg55]IGF-II, which binds to only IGF-IR, also stimulated proteoglycan synthesis in the cells. These findings indicate that IGF-I and IGF-II act as autocrine differentiation factors for this chondrocytic permanent cell line, HCS-2/8, mainly via respective receptors.
Takigawa
M
M
Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Japan. takigawa@dent.okayama-u.ac.jp
Okawa
T
T
Pan
H
H
Aoki
C
C
Takahashi
K
K
Zue
J
J
Suzuki
F
F
Kinoshita
A
A
D000704:Analysis of Variance; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015152:Blotting, Northern; D001859:Bone Neoplasms / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D002356:Cartilage / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D002813:Chondrosarcoma / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D007457:Iodine Radioisotopes; D008297:Male; D008360:Mannosephosphates / Q000494:pharmacology; D011485:Protein Binding; D011509:Proteoglycans / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D017526:Receptor, IGF Type 1 / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D017527:Receptor, IGF Type 2 / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D017077:Culture Media, Conditioned; D007457:Iodine Radioisotopes; D008360:Mannosephosphates; D011509:Proteoglycans; D012333:RNA, Messenger; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1; D002118:Calcium