Human Osteoarthritic Chondrocytes Possess an Increased Number of Insulin‐Like Growth Factor 1 Binding Sites but are Unresponsive to its Stimulation

Abstract

Objective. To characterize the insulin-like growth factor 1 (IGF-1) receptor in human osteoarthritic (OA) and normal adult chondrocytes. The biologic response of chondrocytes to IGF-1 stimulation was examined, as was the presence and synthesis of IGF binding proteins (IGFBP) in these cells.Methods. Binding studies, Northern blot, immunohistochemical analysis, and affinity cross-linking experiments were performed for characterization of the IGF receptor, and the latter method was also used for IGFBP determination. The biologic response was estimated via the incorporation of radiolabeled proline into a newly synthesized protein.Results histochemical studies with a monoclonal antibody (MAb) against the type 1 IGF receptor (aIR3) showed increased staining in OA cartilage compared with normal tissue. Biologic responses of chondrocytes after IGF-1 stimulation revealed that OA chondrocytes were unresponsive, whereas a 2.5-fold increase in new protein synthesis was observed in normal cells. Competition studies in normal chondrocytes revealed that both IGF-1 and IGF-2 displaced radiolabeled IGF-1 in a comparable manner; however, insulin at high concentration weakly competes. Moreover, MAb aIR3 effectively blocked specific binding in normal chondrocytes (77 %), but not in OA chondrocytes (26%). Northern blot and covalent cross-linking analyses revealed the specific band characteristic of type 1 receptor. With the latter technique, other bands corresponding to the IGFBPs were also detected. Comparison between normal and OA chondrocytes showed increased intensity of the IGFBP bands, particularly those corresponding to the IGFBP-3 doublet.Conclusion. It is shown that type 1 IGF receptor is expressed in human articular cartilage and that the level of binding sites is significantly increased in OA chondrocytes. Interestingly, despite the higher level of binding sites in OA cells, no response to IGF-1 stimulation was found in these cells. Our data suggest that this increase in specific binding may involve not only the type 1 IGF receptor but also IGFBP on the cell surface. The latter, by binding the IGF-1, will diminish the bioavailability of IGF-1 and thus prevent its anabolic action.Osteoarthritis (OA) is the most common of the various arthritic disorders affecting humans. The dis-

Normal expression of type 1 insulin‐like growth factor receptor by human osteoarthritic chondrocytes with increased expression and synthesis of insulin‐like growth factor binding proteins

“…This was shown both in vitro, where IGF-1 was found to reduce interleukin-1 (IL-1)-stimulated cartilage degradation (6), and in vivo in an experimental model of OA, in which therapeutically administered IGF-1 in combination with a synthetic protease inhibitor (sodium pentosan polysulfate [PPS]) produced significant improvement in several OA parameters compared with PPS alone (7). IGF-1 alone had no effect, and the IGF-1-treated cartilage was macroscopically similar to the untreated OA cartilage (7), which is consistent with previous data showing that arthritic chondrocytes are hyporesponsive to IGF-1 stimulation (8,9). The exact reason for the improvement when PPS and IGF-1 were administered together is presently unknown.…”

“…Cartilage specimens were obtained under aseptic conditions, and samples were dissected on ice. Chondrocytes were released from articular cartilage by sequential enzymatic digestion at 37"C, as previously described (8). After being released, the cells were centrifuged, washed, seeded at high density (-lo5 cells/cm') and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum (HyClone, Logan, UT) and an antibiotic mixture (100 units/ml of penicillin base and 100 pg/ml of streptomycin base; Gibco BRL) at 37°C in a humidified atmosphere of 5% COz, 95% air.…”

“…Total RNA was isolated from confluent cultures of primary normal and OA human chondrocytes, as previously described (8). Briefly, the cells were lysed in a preheated (60°C) buffer containing 20 mM sodium acetate, pH 5,0.5% SDS, and 1 mM EDTA.…”

“…Evidence is mounting which suggests that the aborted reparation of the damaged cartilage, as seen in OA, may have to do more with chondrocyte hyporesponsiveness to growth factors than to their synthesis. This hypothesis appears to apply to IGF-1, since although OA chondrocytes synthesized more IGF-1 than normal chondrocytes (30,31) and presented an increased number of IGF-1 binding sites per cell, these diseased cells were not responsive to IGF-1 stimulation (8).…”

Nitric oxide inhibition of IGF‐1 stimulated proteoglycan synthesis: Role of cGMP

“…In healthy cartilage, it induces expression of collagen type I1 and proteoglycan core protein, thus stabilizing the chondrocyte phenotype, and stimulates synthesis of collagen and proteoglycans [29]. However, chondrocytes in arthritic cartilage of humans [9] and animals [29] have decreased anabolic responses to IGF-1.…”