Cartilage is a primary target tissue for the IGFs. The mitogenic activity of these peptides is regulated by a family of high-affinity IGF-binding proteins (IGFBP-1 to -6). We characterized the IGFBPs produced by cultured chondrocytes derived from rib cartilage of prepubertal rabbits. Culture medium, which had been conditioned by these cells for 48 h showed bands of 22 kDa, 24 kDa and a 31/32 kDa doublet by Western ligand blotting with [(125)I]IGF-II. When the cells were grown in the presence of increasing amounts of IGF-I or IGF-II, the 31/32 kDa doublet increased in intensity (reaching a plateau of about 11-fold stimulation between 2 and 10 nM IGF-I). The 22 kDa and 24 kDa bands increased only slightly while a 26 kDa band became faintly visible. By Western immunoblotting the 31/32 kDa doublet was identified as IGFBP-5. An IGF-I analog with reduced affinity for IGFBPs, Long-R3 IGF-I, also induced IGFBP-5, while insulin was less effective (2.2-fold stimulation at 10 nM). IGF-I protected IGFBP-5 against proteolytic degradation by conditioned medium. IGF-I also enhanced the level of IGFBP-5 mRNA. LY294002, a specific inhibitor of the intracellular signaling molecule phosphatidylinositol 3-kinase, inhibited stimulation of IGFBP-5 by IGF-I. Dexamethasone suppressed IGFBP-5 (by 70% at 20 nM) but, at the same time, a 39/41 kDa doublet (presumably IGFBP-3) was induced. IGFBP-5 has been shown in several cell types to enhance the mitogenic activity of IGF-I. IGFBP-3 generally acts as a growth inhibitor. Therefore, the differential effects of dexamethasone on these regulatory proteins could account, at least in part, for the growth-arresting effect of this glucocorticoid.

Partial proteolysis of insulin-like growth factor-binding protein-3 (IGFBP-3) lowers its affinity for IGFs. Presumably, this leads to destabilization of the ternary IGF-IGFBP-3-acid-labile subunit complex in the circulation and an increased bioavailability of IGFs. We investigated the effect of GH on IGFBP-3 proteolysis by comparing serum from normal mice and GH-deficient dwarf mice. While normal mouse serum degraded 125I-IGFBP-3, this activity declined with age. In contrast, serum from dwarf mice displayed strong proteolytic activity at all ages tested (up to 10 weeks). In dwarf mice of 4 weeks and older, this activity could not be inhibited by EDTA and 1,10-phenanthroline, indicating the presence of a divalent cation-independent protease. Prolonged treatment with GH (4 weeks) did not decrease the overall potency of the serum to degrade IGFBP-3, but partially restored the ability of EDTA to inhibit IGFBP-3 protease activity. GH deficiency therefore appears to induce a new kind of IGFBP-3 protease. Similarly, serum from hypophysectomized rats displayed enhanced IGFBP-3 protease activity compared with control rat serum. These results suggest that a protease induced under conditions of severe GH deficiency may contribute to making IGFs optimally available to the tissues.

High (pharmacological) doses of glucocorticoids inhibit the proliferation of growth plate chondrocytes, which leads to one of the side-effects of these steroids, namely suppression of longitudinal growth. Growth inhibition by glucocorticoids is thought to be mediated in part by impaired action of components of the IGF axis, which are important for chondrocyte regulation and hence for longitudinal growth. The aim of the present study was to determine whether glucocorticoid-induced growth retardation involves changes in IGF axis components. Chondrocytes were isolated from epiphyseal growth plates of neonatal piglets and treated with pharmacological doses of dexamethasone (DXM) for 24 h to study glucocorticoid-induced growth retardation. Under IGF-I-supplemented (10 nM) culture conditions, IGF-binding proteins (IGFBPs)-2, -4 and -5 were secreted by the growth plate chondrocytes and IGFBP-2 protein and mRNA levels were decreased by the DXM treatment, whereas IGFBP-4 and -5 were not affected. Proliferation of the chondrocytes, as measured by [(3)H]thymidine incorporation, was 3.5-fold higher in serum-supplemented medium in contrast to IGF-I-supplemented (10 nM) medium. In the presence of serum, DNA synthesis was significantly inhibited by 50-63% when treated with 100 nM DXM, which was prevented by the glucocorticoid-receptor antagonist Org34116. mRNA levels of IGF axis components were determined using Northern blot analysis. IGFBP-2 to -6 were expressed in the chondrocytes, IGFBP-1 was absent and both IGF-I and IGF-II, and the type I and type II IGF receptors were expressed. Treatment with DXM (100 nM) resulted in a 2-fold increase in mRNA levels of both IGFBP-5 and the type I IGF receptor, whereas IGFBP-2 mRNA levels decreased by 55%, in concert with the decrease in protein level observed under IGF-I-supplemented culture conditions. The changes in mRNA levels due to the DXM treatment were prevented by the glucocorticoid receptor antagonist. Our data show that exposure to pharmacological doses of DXM results in inhibition of proliferation and changes in components of the IGF axis, IGFBP-2 and -5 and the type I IGF receptor, suggesting a role for these components in glucocorticoid-induced growth retardation at the local level of the growth plate.

Glucocorticoid (GC) treatment in childhood can lead to suppression of longitudinal growth as a side effect. The actions of GCs are thought to be mediated in part by impaired action of the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins (IGFBP-1 to -6). We have studied the effects of GCs on IGF and IGFBP expression at the local level of the growth plate, using non-radioactive in situ hybridization. We treated 3-week-old normal mice for 4 weeks with dexamethasone (DXM). We also treated human IGF-II (hIGF-II) transgenic mice in order to investigate whether IGF-II could protect against the growth retarding effect of this GC. DXM treatment resulted in general growth retardation in both mice strains, however, only in normal mice was tibial length decreased. In both normal and hIGF-II trangenic mice, the total width of the growth plate was not affected, whereas the width of the proliferative zone decreased as a result of the DXM treatment. Additionally, only in normal mice, the width of the hypertrophic zone thickened. Only expression of IGF-I, IGF-II and IGFBP-2 could be detected in the growth plates of 7-week-old normal mice. IGFBP-1, -3, -4, -5 and -6 mRNAs were not detected. DXM treatment of normal mice induced a significant 2.4-fold increase in the number of cells expressing IGF-I mRNA, whereas IGF-II and IGFBP-2 mRNA levels were not affected. In hIGF-II transgenic mice, IGF-I mRNA levels were significantly increased, while endogenous IGF-II and IGFBP-2 mRNAs were unaffected, compared to normal animals. DXM treatment of the hIGF-II transgenic mice induced a further increase of IGF-I mRNA expression, to a similar extent as in DXM-treated normal mice. The increase of IGF-I due to DXM treatment in normal mice might be a reaction in order to minimize the GC-induced growth retardation. Another possibility could be that the increase of IGF-I would contribute to the GC-induced growth retardation by accelerating the differentiation of chondrocytes, resulting in accelerated ossification. In the growth plates of hIGF-II transgenic mice, the higher basal level of IGF-I, might be responsible for the observed partial protection against the adverse effects of GCs on bone.

The insulin-like growth factor (IGF) system is an important mediator of postnatal longitudinal growth, and the growth inhibiting effects of glucocorticoid (GC) treatment are suggested to be due to impaired action of the IGF system. However, the precise changes of the IGFs and the IGF-binding proteins (IGFBPs) in the growth plate, occurring upon short-term GC treatment have not been characterized. Prepubertal mice treated daily with dexamethasone (DXM) for 7 days, showed significant growth inhibition of total body length and weight and weight of the liver, thymus and spleen, whereas the weight of the kidneys was not affected. Analysis of the tibial growth plate showed that the total growth plate width significantly decreased to 84.5% of control values, caused by a significant decrease in the proliferative zone. The number of proliferating cell nuclear antigen (PCNA)-positive chondrocytes in the proliferative zone decreased significantly (to 40%) and TUNEL staining showed a significant 1.6-fold increase in apoptotic hypertrophic chondrocytes. In the growth plates, both IGF-I and IGF-II, as well as IGFBP-2 mRNAs were detected, mainly in the proliferative and prehypertrophic zones. DXM treatment significantly decreased the number of chondrocytes expressing IGF-I, whereas the number of chondrocytes expressing IGF-II and IGFBP-2 were not affected. The decrease in IGF-I expression in the growth plate indicates that GC treatment affects IGF-I at the local level of the growth plate, which could contribute to the GC-induced growth retardation.