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Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat
M C Slootweg1, C Ohlsson, J P Salles
1Research Institute for Endocrinology, Free University Hospital, Amsterdam, The Netherlands.
Abstract:
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)
Insights
Insulin-like growth factors (IGFs) I and II decrease growth hormone (GH) receptor binding and mRNA levels in osteoblasts. Conversely, IGF binding proteins (IGFBPs) 2 and 3 enhance GH receptor binding and mRNA, showing opposite regulatory roles.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Growth hormone (GH) mediates biological actions via its receptor on osteoblasts.
- Serum components modulate GH receptor binding, with fetal calf serum decreasing it.
- Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) were investigated for their roles in modulating GH receptor activity.
Purpose of the Study:
- To investigate the modulatory effects of IGF-I, IGF-II, IGFBP-2, and IGFBP-3 on GH receptor binding and expression in osteoblasts.
- To determine the impact of these factors on GH receptor mRNA levels.
- To assess the mitogenic effects of IGFs and IGFBPs on osteosarcoma cells and their potentiation of GH-induced mitogenesis.
Main Methods:
- Radioligand binding assays using [125I]hGH to measure GH receptor binding.
- Solution hybridization ribonuclease protection assays to quantify GH receptor mRNA levels.
- Cell proliferation assays to assess mitogenic activity.
Main Results:
- IGF-I and IGF-II decreased [125I]hGH binding and GH receptor mRNA levels.
- IGFBP-2 and IGFBP-3 increased [125I]hGH binding, with IGFBP-2 also increasing GH receptor mRNA levels.
- IGFBP-2 and IGFBP-3 demonstrated mitogenic effects on osteosarcoma cells and potentiated GH-induced mitogenesis at low GH concentrations.
Conclusions:
- IGF-I and IGF-II exert opposite effects to IGFBP-2 and IGFBP-3 on GH receptor binding.
- Regulation of the GH receptor occurs, at least partly, at the mRNA level, with potential IGF-independent effects from IGFBPs.
- IGFBP-2 and IGFBP-3 possess mitogenic properties and can potentiate GH-induced proliferation in osteosarcoma cells.