The growth potentiating effects of the insulin-like growth factor (IGF)-I and IGF-II are modulated by a family of six insulin-like growth factor binding proteins (IGFBPs). Despite the similarity in amino acid sequences of the IGFBPs, their effects on the growth of bone cells differ. Studies on the molecular mechanisms for IGFBP-4 actions revealed that coincubation of bone cells with IGFBP-4 and 125I-IGF-I or 125I-IGF-II decreased the binding of both of these ligands in a dose-dependent manner. In addition, IGFBP-4 decreased the binding of IGF-I tracer to purified type I IGF receptor. These data in conjunction with data showing that IGFBP-4 had no effect on cell proliferation induced by analogs of IGF-I or IGF-II, which exhibited > 100-fold reduced affinity for binding to IGFBP-4 suggest that IGFBP-4 may inhibit IGF action by preventing the binding of ligand to its membrane receptor. In contrast to IGFBP-4, IGFBP-5 treatment increased the binding of IGF tracer to bone cells but did not increase the binding of 125I-IGF-I to type I IGF receptor. Studies on the mechanism by which IGFBP-5 increased the binding of 125I-IGF tracer to bone cells suggest that IGFBP-5 could facilitate IGF binding by a mechanism in which IGFBP-5 has cell surface binding sites independent of IGF receptors. These data in conjunction with the findings that IGFBP-5 potentiated cell proliferation even in the presence of those same IGF analogs that exhibited > 200-fold reduced affinity for binding to IGFBP-5, suggest that IGFBP-5 may in part stimulate bone cell proliferation by an IGF-independent mechanism involving IGFBP-5-specific cell surface binding sites.

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.

Teratocarcinoma cells provide us with a model system for the study of differentiation and development. One of the best characterized cell lines, the embryonal carcinoma stem cell line F9, differentiates after treatment with retinoic acid (RA) and dibutyryl cyclic AMP into parietal endoderm. This differentiation process is accompanied by the induction of several genes, for example, those encoding collagen IV, plasminogen activator and intermediate filaments like laminin. In contrast, a marked reduction of stable messenger RNA has been observed for the gene encoding p53 and for c-myc. Both cellular oncogenes seem to be involved in the regulation of cellular proliferation and neoplastic transformation. For growth-arrested 3T3 fibroblasts, growth-factor-induced changes of myc RNA are controlled at the level of transcription. In contrast, F9 cells provide a differentiation system in which cells are able to change from a tumorigenic state into non-dividing, non-tumorigenic endodermal cells. The latter process enabled us to study the regulation of myc and p53 genes in the same cells at different stages of growth, tumorigenicity and differentiation. Here we report that down-regulation of stable myc and p53 RNA during irreversible differentiation of F9 cells occurs at the post-transcriptional level. Using an in vitro nuclear transcription assay, we found that the polymerase II density on both genes remains constant during differentiation. In agreement with this interpretation, we detected myc RNA as stable transcripts in differentiated F9 cells after treatment of the cells with cycloheximide. The post-transcriptional regulatory mechanisms controlling p53 and myc stability follow different kinetics. Whereas the down-regulation of myc seems to be an early event of F9 differentiation occurring within the first 24 h, the post-transcriptional regulation of p53 occurs at a later stage (two to three days), possibly as a consequence of cell cycle changes.

We investigated the regulation of Sox9, a transcription factor known to play a role in chondrogenesis, by bone morphogenetic protein-2 (BMP-2) and hedgehog proteins in order to better understand their signaling function in endochondral bone formation. The mesenchymal progenitor cell line C3H10T1/2 was stimulated with BMP-2. Sox9 expression levels were measured by quantitative reverse transcriptase-polymerase chain reaction and Northern analysis. We found that Sox9 was up-regulated by BMP-2 in a dose-dependent manner. The expression of Col2a1, a downstream response gene of Sox9, was also significantly increased upon BMP-2 addition. We also monitored Sox9 expression after the addition of BMP-2 to osteosarcoma cell lines; BMP-2 treatment increased Sox9 mRNA levels in MG63, considered to be early osteoblast-like, but not in human osteogenic sarcoma (HOS) cells, which are thought to be more advanced in the osteoblastic lineage. This response seems to be influenced by differences in BMP receptor expression; MG63 cells express BMP receptor IA (BMPR-IA), whereas HOS cells express BMPR-IA and BMPR-IB. We also saw an increase in Sox9 mRNA levels in BMP-2-treated primary human bone cells (HBCs) derived from femoral heads. We found that in addition to BMP-2, Sonic and Indian hedgehog can increase Sox9 expression in C3H10T1/2 and primary HBCs. Time course studies with C3H10T1/2 cells after BMP-2 stimulation showed increasing expression of cartilage markers, decrease of collagen I mRNA, and a late induction of osteocalcin expression. Moreover, the treatment of C3H10T1/2 cells with Sox9 antisense oligonucleotides revealed that Sox9 is a downstream mediator of BMP-2 affecting the expression of chondrocyte and osteoblast marker genes. Our data show that Sox9 is an important downstream mediator of the BMP-2 and hedgehog signaling pathways in osteogenic