Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells.
GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)
Slootweg
M C
MC
Research Institute for Endocrinology, Free University Hospital, Amsterdam, The Netherlands.
Ohlsson
C
C
Salles
J P
JP
de Vries
C P
CP
Netelenbos
J C
JC
D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D013006:Growth Hormone / Q000378:metabolism; D018971:Insulin-Like Growth Factor Binding Protein 2; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D012516:Osteosarcoma / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018971:Insulin-Like Growth Factor Binding Protein 2; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D011986:Receptors, Somatotropin; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D013006:Growth Hormone