Effects of IGF-I, EGF, and FGF on proteoglycans synthesized by fractionated chondrocytes of rat rib growth plate

The effects of insulin-like growth factor (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF), or 10% newborn calf serum (NCS) on the amount and structure of the proteoglycans synthesized by fractionated chondrocytes from rat growth plate were investigated. Proliferative cells (fraction II) or resting cells (fraction III) synthesized more proteoglycans than hypertrophic cells (fraction I). Addition of IGF-I to the cultures increased proteoglycan synthesis more than addition of EGF or FGF. EGF and FGF induced synthesis of proteoglycans of smaller molecular size with a lower proportion of aggregates. The size of the constituent glycosaminoglycan chains did not differ between control and growth factor-treated cells. The present study demonstrates that proteoglycan structure and synthesis are modified by growth factors to different extents, depending on the maturation stage of the target cells.     

Co-ordination of TGF-beta and FGF signaling pathways in bone organ cultures

Transforming growth factor-beta (TGF-beta) is known to regulate chondrocyte proliferation and hypertrophic differentiation in embryonic bone cultures by a perichondrium dependent mechanism. To begin to determine which factors in the perichondrium mediate the effects of TGF-beta, we studied the effect of Insulin-like Growth Factor-1 (IGF-I) and Fibroblast Growth Factors-2 and -18 (FGF2, FGF18) on metatarsal organ cultures. An increase in chondrocyte proliferation and hypertrophic differentiation was observed after treatment with IGF-I. A similar effect was seen after the perichondrium was stripped from the metatarsals suggesting IGF-I acts directly on the chondrocytes. Treatment with FGF-2 or FGF-18 resulted in a decrease in bone elongation as well as hypertrophic differentiation. Treatment also resulted in a decrease in BrdU incorporation into chondrocytes and an increase in BrdU incorporation in perichondrial cells, similar to what is seen after treatment with TGF-beta1. A similar effect was seen with FGF2 after the perichondrium was stripped suggesting that, unlike TGF-beta, FGF2 acts directly on chondrocytes to regulate proliferation and hypertrophic differentiation. To test the hypothesis that TGF-beta regulates IGF or FGF signaling, activation of the receptors was characterized after treatment with TGF-beta. Activation was measured as the level of tyrosine phosphorylation on the receptor. Treatment with TGF-beta for 24h did not alter the level of IGFR-I tyrosine phosphorylation. In contrast, treatment with TGF-beta resulted in and increase in tyrosine phosphorylation on FGFR3 without alterations in total FGFR3 levels. TGF-beta also stimulated expression of FGF18 mRNA in the cultures and the effects of TGF-beta on metatarsal development were blocked or partially blocked by pretreatment with FGF signaling inhibitors. The results suggest a model in which FGF through FGFR3 mediates some of the effects of TGF-beta on embryonic bone formation.     

Transforming growth factor-beta,basic fibroblast growth factor,and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells

Transforming growth factor beta-1 (1GF-β) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serumcontaining medium by 58%. The effect of TGF-β on cell proliferation in serumfree medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-β inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-β in serum-free medium was not different than TGF-β alone. TGF-β depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-β did not affect proliferation. TGF-β inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-β and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-β and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-β interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-β on proliferation of clonally derived porcine satelite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF. © 1993 Wiley-Liss, Inc.     

Growth factor regulation of human growth plate chondrocyte proliferation in vitro

Linear growth occurs as the result of growth plate chondrocytes undergoing proliferative and hypertrophic phases. Paracrine feedback loops that regulate the entry of chondrocytes into the hypertrophic phase have been shown and similar pathways likely exist for the proliferative phase. Human long-bone growth plate chondrocytes were cultured in vitro. The proliferative effects of a variety of factors were determined by [3H]thymidine uptake and the gene expression profile of these cells was determined by DNA microarray analysis. Serum, insulin-like growth factor (IGF)-I and -II, transforming growth factor-beta (TGF-beta, fibroblast growth factor (FGF)-1, -2, and -18, and platelet-derived growth factor (PDGF)-BB were potent stimulators of proliferation. FGF-10, testosterone, and bone morphogenetic proteins (BMP)-2, -4, and -6 inhibited proliferation. Microarray analysis showed that the genes for multiple members of the IGF-I, TGF-beta, FGF, and BMP pathways were expressed, suggesting the presence of autocrine/paracrine pathways that regulate the proliferative phase of growth plate-mediated growth.     

A comparison of the responses of cultured myoblasts and chondrocytes to fibroblast and epidermal growth factors.

The effects of fibroblast and epidermal growth factors on proliferation and differentiation of cultured myoblasts and chondrocytes have been compared. FGF stimulated myoblast proliferation, as determined by monitoring levels of DNA synthesis during seven days growth in vitro and by the morphology of the cultures after myotube formation. EGF has relatively little effect on myoblast proliferation. With chondrocytes, both FGF and EGF are mitogenic and FGF's, but not EGF's effect is potentiated by dexamethasone. One implication of these results is that in the course of differentiation cell types which develop from the same embryonic origin as fibroblasts are controlled by different sets of mitogenic factors. Myoblasts become primarily dependent on mitogenic agents such as FGF while chondrocytes can respond to both FGF and EGF.     

PDGF BB stimulates proliferation and differentiation in cultured chondrocytes from rat rib growth plate.

The role of two isoforms (PDGF AA and PDGF BB) of platelet derived growth factor either alone or in combination with insulin-like growth factor I, on the regulation of proliferation and differentiation of rat rib growth plate chondrocytes was analyzed. PDGF BB increased DNA-synthesis in a dose dependent manner with a half maximal effect at 1 ng/ml. When PDGF BB was combined with IGF-I, an additive effect on DNA-synthesis was observed. PDGF AA and BB alone or combined with IGF-I had no appreciable effects on proteoglycan synthesis. Both homodimers caused an increase in AP-activity, indicating stimulation of cell differentiation. Cultured chondrocytes bound 125I-PDGF AA and 125I-PDGF BB and after stimulation with PDGF expressed c-fos protein. Thus, both homodimers play an important role in chondrocyte differentiation and together with IGF-I interact in the regulation of longitudinal bone growth.     

Binding of insulin-like growth factor-I (IGF-I) to primary cultures of chondrocytes from rat rib growth cartilage

Binding of insulin-like growth factor I (IGF-I) to cultured resting, proliferative and hypertrophic growth plate chondrocytes was investigated. The optimal binding conditions and the extent of degradation of the 125I-IGF-I at 20 degrees C were analyzed in a time-course study. The maximal binding without noticeable degradation was observed after 3 h. The binding of IGF-I to the proliferative cells was 2-fold higher than to the resting and the hypertrophic cells. On the proliferative chondrocytes two classes of receptors with different affinities were found. 125I-IGF-I could be displaced from the proliferative cells by unlabelled IGF-I, IGF-II and insulin, respectively. Half maximal binding was observed at 0.3 nmol/l (= 2.2 micrograms/l) of IGF-I, 4.3 nmol/l (= 32 micrograms/l) of IGF-II and 350 nmol/l (= 2000 micrograms/l) of insulin. No specific binding of human growth hormone (hGH) could be demonstrated. When binding of epidermal growth factor (EGF) to the proliferative cells was assessed, little, but specific binding was observed.     

Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with various combinations of transforming growth factor (TGF)-beta, fibroblast growth factor (FGF), and insulin-like growth factor I (IGF-I). In serum-free defined medium the following observations were made: TGF-beta depressed proliferation and inhibited differentiation; FGF stimulated proliferation and depressed differentiation; IGF-I stimulated proliferation to a small degree but demonstrated a more pronounced stimulation of differentiation. In evaluating combinations of these three factors, the differentiation inhibiting effect of TGF-beta could not be counteracted by any combination of IGF-I or FGF. The proliferation-depressing activity of TGF-beta, however, could not inhibit the mitogenic activity of FGF. Maximum stimulation of proliferation was observed in the presence of both FGF and IGF-I. The highest percentage fusion was also observed under these conditions, but differentiation with minimal proliferation resulted from treatment with IGF-I, alone. By altering the concentrations of TGF-beta, FGF, and IGF-I, satellite cells can be induced to proliferate, differentiate, or to remain quiescent.     

FGF18 is required for early chondrocyte proliferation, hypertrophy and vascular invasion of the growth plate

Fibroblast growth factor 18 (FGF18) has been shown to regulate chondrocyte proliferation and differentiation by signaling through FGF receptor 3 (FGFR3) and to regulate osteogenesis by signaling through other FGFRs. Fgf18(-/-) mice have an apparent delay in skeletal mineralization that is not seen in Fgfr3(-/-) mice. However, this delay in mineralization could not be simply explained by FGF18 signaling to osteoblasts. Here we show that delayed mineralization in Fgf18(-/-) mice was closely associated with delayed initiation of chondrocyte hypertrophy, decreased proliferation at early stages of chondrogenesis, delayed skeletal vascularization and delayed osteoclast and osteoblast recruitment to the growth plate. We further show that FGF18 is necessary for Vegf expression in hypertrophic chondrocytes and the perichondrium and is sufficient to induce Vegf expression in skeletal explants. These findings support a model in which FGF18 regulates skeletal vascularization and subsequent recruitment of osteoblasts/osteoclasts through regulation of early stages of chondrogenesis and VEGF expression. FGF18 thus coordinates neovascularization of the growth plate with chondrocyte and osteoblast growth and differentiation.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

Interaction of FGF,Ihh/Pthlh,and BMP signaling integrates chondrocyte proliferation and hypertrophic differentiation

Mutations in fibroblast growth factor (FGF) receptor 3 lead to the human dwarfism syndrome achondroplasia. Using a limb culture system, we have analyzed the role of FGF signaling and its interaction with the Ihh/Pthlh and BMP pathways in regulating chondrocyte differentiation. In contrast to previous suggestions, we demonstrate that FGF signaling accelerates both the onset and the pace of hypertrophic differentiation. We furthermore found that FGF and BMP signaling act in an antagonistic relationship regulating chondrocyte proliferation, Ihh expression, and the process of hypertrophic differentiation. Importantly, BMP signaling rescues the reduced domains of proliferating and hypertrophic chondrocytes in a mouse model for achondroplasia. We propose a model in which the balance of BMP and FGF signaling adjusts the pace of the differentiation process to the proliferation rate.     

Immunohistochemical analysis of EGF in epiphyseal growth plate from normal,hypophysectomized, and growth hormone-treated hypophysectomized rats

Epiphyseal growth plate cartilages from the proximal tibia of normal, hypophysectomized, and growth hormone (GH)-treated hypophysectomized rats were subjected to immunohistochemistry for detection of epidermal growth factor (EGF). In the normal growth plate, EGF was distributed mainly in the proliferative zone. Hypophysectomy resulted in considerable atrophy of the chondrocytes and the cartilage matrix (a decreased number of mature-type chondrocytes and a decreased ratio of proliferating to hypertrophic chondrocytes) and a significant diminution of EGF immunoreactivity. Treatment with GH reversed these effects of hypophysectomy, causing an increased thickness of the growth plate and EGF-reactive sites in all chondrocyte layers. The most intense immunostaining for EGF, however, was frequently seen in the nuclei of chondrocytes with flattened appearance. It appears that EGF could be incorporated or synthesized in chondrocytes having marked mitogenic activity. The present results, taken with previous data on EGF involvement in growth of cartilaginous tissue in vivo and in vitro, strongly suggest that EGF-immunoreactive chondrocytes are involved in cartilage proliferation and growth under the specific influence of GH.     

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

FGF upregulates osteopontin in epiphyseal growth plate chondrocytes: Implications for endochondral ossification

Fibroblast growth factor receptor 3 (FGFR3) signaling pathways are essential for normal longitudinal bone growth. Mutations in this receptor lead to various human growth disorders, including Achondroplasia, disproportionately short-limbed dwarfism, characterized by narrowing of the hypertrophic region of the epiphyseal growth plates. Here we find that FGF9, a preferred ligand for FGFR3 rapidly induces the upregulation and secretion of the matrix resident phosphoprotein, osteopontin (OPN) in cultured chicken chondrocytes. This effect was observed as early as two hours post stimulation and at FGF9 concentrations as low as 1.25 ng/ml at both mRNA and protein levels. OPN expression is known to be associated with chondrocyte and osteoblast differentiation and osteoclast activation. Unexpectedly, FGF9 induced OPN was accompanied by inhibition of differentiation and increased proliferation of the treated chondrocytes. Moreover, FGF9 stimulated OPN expression irrespective of the differentiation stage of the cells or culture conditions. In situ hybridization analysis of epiphyseal growth plates from chicken or mice homozygous for the Achondroplasia, G369C/mFGFR3 mutation demonstrated co-localization of OPN expression and osteoclast activity, as evidenced by tartarate resistant acid phosphatase positive cells in the osteochondral junction. We propose that FGF signaling directly activates OPN expression independent of chondrocytes differentiation. This may enhance the recruitment and activation of osteoclasts, and increase in cartilage resorption and remodeling in the chondro-osseus border.     

Mechanoregulation of chondrocyte proliferation, maturation, and hypertrophy: ion-channel dependent transduction of matrix deformation signals

Mechanical stress-induced matrix deformation plays a fundamental role in regulating cellular activities; however, little is known about its underlying mechanisms. To understand the effects of matrix deformation on chondrocytes, we characterized primary chondrocytes cultured on three-dimensional collagen scaffoldings, which can be loaded mechanically with a computer-controlled "Bio-Stretch" device. Cyclic matrix deformation greatly stimulated proliferation of immature chondrocytes, but not that of hypertrophic chondrocytes. This indicates that mechanical stimulation of chondrocyte proliferation is developmental stage specific. Synthesis of cartilage matrix protein (CMP/matrilin-1), a mature chondrocyte marker, and type X collagen, a hypertrophic chondrocyte marker, was up-regulated by stretch-induced matrix deformation. Therefore, genes of CMP and type X collagen are responsive to mechanical stress. Mechanical stimulation of the mRNA levels of CMP and type X collagen occurred exactly at the same time points when these markers were synthesized by nonloading cells. This indicates that cyclic matrix deformation does not alter the speed of differentiation, but affects the extent of differentiation. The addition of the stretch-activated channel blocker gadolinium during loading abolished mechanical stimulation of chondrocyte proliferation, but did not affect the up-regulation of CMP mRNA by mechanical stretch. In contrast, the calcium channel blocker nifedipine inhibited both the stretch-induced proliferation and the increase of CMP mRNA. This suggests that stretch-induced matrix deformation regulates chondrocyte proliferation and differentiation via two signal transduction pathways, with stretch-activated channels involved in transducing the proliferative signals and calcium channels involved in transducing the signals for both proliferation and differentiation.     

Somatomedins (insulin-like growth factors), but not growth hormone, are mitogenic for chicken heart mesenchymal cells and act synergistically with epidermal growth factor and brain fibroblast growth factor

Chicken, ovine or human growth hormones have no mitogenic effect on chicken heart mesenchymal cells, which are proliferatively quiescent at low culture densities in medium containing heparinized, heat-defibrinogenated rooster plasma at 10%. Sm-C/IGF-I (15 ng/ml; 2 nM), MSA/rIGF-II (50 ng/ml; 7 nM), insulin (10,000 ng/ml; 1750 nM) or proinsulin (16,000 ng/ml; 1750 nM), however, cause these cells to increase threefold in number during four days of incubation. While EGF alone at 100 ng/ml causes threefold multiplication at four days and brain FGF causes a sixfold increase, EGF acts synergistically with Sm-C/IGF-I, MSA/rIGF-II, insulin or proinsulin to cause 18-fold multiplication, and brain FGF acts synergistically with IGFs to cause 20-fold multiplication. EGF and brain FGF, however, show no mitogenic synergy. Addition to the plasma-containing culture medium of a monoclonal antibody to Sm-C/IGF-I nearly abolishes the mitogenic effect of added EGF or brain FGF but does not affect the autonomous (mitogenic hormone-independent) proliferation of RSV-infected chicken heart mesenchymal cells. These findings support the somatomedin hypothesis for growth hormone action and suggest that potentiation of the activity of other mitogenic hormones, like EGF and FGF, makes a significant contribution to control of cell proliferation by the GH/IGF axis.     

Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes

Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition.     

Antiproliferative effects of insulin-like growth factor-binding protein-3 in mesenchymal chondrogenic cell line RCJ3.1C5.18. relationship to differentiation stage

Chondrogenesis results from a complex equilibrium between chondrocyte proliferation and differentiation. Insulin-like growth factors (IGFs) have a crucial role in chondrogenesis, but their mechanisms of action are not well defined. IGF-binding protein-3 (IGFBP-3) is the major carrier for circulating IGFs in postnatal life, and has been shown to have IGF-independent effects on proliferation of several cancer cell lines. In this study, we have evaluated the IGF-independent and -dependent effects of IGFBP-3 on chondrocyte proliferation and the relationship of these effects with chondrocyte differentiation stage. We used the RCJ3.1C5.18 nontransformed mesenchymal chondrogenic cell line, which, over 2 weeks of culture, progresses through the differentiation pathway exhibited by chondrocytes in the growth plate. We demonstrated that IGFBP-3 inhibited, in a dose-dependent manner (1-30 nm), the proliferation of chondroprogenitors and early differentiated chondrocytes, stimulated by des-(1-3)-IGF-I and longR(3)-IGF-I (IGF-I analogs with reduced affinity for IGFBP-3), and by insulin and IGF-I. In terminally differentiated chondrocytes, IGFBP-3 retained the ability to inhibit cell proliferation stimulated by IGF-I, but had no effect on cell growth stimulated by insulin, or des-(1-3)-IGF-I or longR(3)IGF-I. By monolayer affinity cross-linking, we demonstrated a specific IGFBP-3-associated cell-membrane protein of approximately 20 kDa. We determined that IGFBP-3 has an antiproliferative effect on chondrocytes and, that this effect is related to the differentiation process. In chondroprogenitors and early differentiated chondrocytes, antiproliferative effect of IGFBP-3 is mainly IGF-independent, whereas, following terminal differentiation this effect is IGF-dependent.