Stimulated Proteoglycan Synthesis Research Articles

Stimulation of proteoglycan synthesis in explants of porcine articular cartilage by recombinant osteogenic protein-1 (bone morphogenetic protein-7).

Osteogenic protein-1 (also known as bone morphogenetic protein-7) is a member of the bone morphogenetic protein family. Bone morphogenetic proteins and related members of the TGF-beta (transforming growth factor-beta) superfamily are involved in the development and repair of bone. Recombinant bone morphogenetic proteins induce the formation of new cartilage and bone at heterotopic sites. We investigated the influence of recombinant osteogenic protein-1 (at doses of three, ten, thirty, or 100 nanograms per milliliter) on the synthesis and release of proteoglycans and the maintenance of a steady-state concentration of proteoglycans in explants of porcine articular cartilage that were maintained in chemically defined serum-free medium. We found a dose-dependent stimulation of proteoglycan synthesis and a concurrent decrease in the rate of release of proteoglycans from the explants. The size of the proteoglycan monomers and the composition of the glycosaminoglycan chains in the untreated articular cartilage were similar to those in the articular cartilage treated with osteogenic protein-1. The capacity of the newly synthesized proteoglycan monomers to form aggregates with exogenous hyaluronic acid was found to be similar to that of proteoglycans in bovine nasal cartilage. Our results demonstrated that osteogenic protein-1 stimulated the synthesis of proteoglycans and diminished the release of proteoglycans from explants of porcine articular cartilage. The maintenance and repair of articular cartilage is a formidable challenge in clinical orthopaedics. The stimulation of proteoglycan synthesis by osteogenic protein-1 (bone morphogenetic protein-7) in explants of cartilage maintained in chemically defined serum-free medium implies that recombinant osteogenic protein-1 may play a role in the maintenance of a steady-state concentration of proteoglycans in articular cartilage, a desirable prerequisite for optimum repair of cartilage. Osteogenic protein-1 can initiate the formation of cartilage from mesenchymal cells. Once new cartilage has formed at the site of repair, osteogenic protein-1 also may maintain the synthesis of proteoglycans.

Effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc.

This study is a unique in vitro study on the effects of hydrostatic pressure on human intervertebral disc metabolism. To investigate the effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc. Mechanical stress and hydrostatic pressures influence proteoglycan and protein synthesis rates in bovine articular cartilage and coccygeal discs. However, the mechanism of matrix synthesis regulation of the intervertebral disc under mechanical stress has not been elucidated. Twenty-eight human lumbar intervertebral discs obtained from surgery and from cadavers at autopsy were used. Each tissue fraction was charged with medium in a plastic syringe and placed in a water-filled hydrostatic pressure-control vessel. The hydrostatic pressures applied were 1 (control), 3, and 30 atm (atm = atmospheres) for 2 hours. The proteoglycan and protein synthesis rates were determined by radioisotope incorporation. The production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 were measured by a one-step enzyme immunoassay method using monoclonal antibodies. Three atm pressure stimulated proteoglycan synthesis rates in the nucleus pulposus and inner anulus (n = 14 in each tissue). Compared with the control group, 30 atm pressure significantly inhibited proteoglycan synthesis in the inner anulus (P = 0.011). In the nucleus pulposus, matrix metalloproteinase-3 production was stimulated at a pressure of 30 atm relative to 3 atm (P = 0.014, n = 16 in each tissue). The highest tissue inhibitor of metalloproteinases-1 production showed highest values at 3 atm pressure in the inner anulus (n = 16 in each tissue). The results suggest that hydrostatic pressure influences intervertebral disc cell metabolism. A physiologic level of hydrostatic pressure (3 atm) may act as an anabolic factor for stimulation of proteoglycan synthesis and tissue inhibitor of metalloproteinases-1 production. This may be essential for maintaining the matrix of the disc. If the pressure was 30 atm or more or 1 atm or less, a catabolic effect will be predominant, with reduction of proteoglycan synthesis rate and increase of matrix metalloproteinase-3 production. Abnormal hydrostatic pressure, therefore, may accelerate disc degeneration.

Injury-induced alterations in newly synthesized sulphated proteoglycans from rabbit arterial neointima covered by regenerated endothelium.

Proteoglycan (PG) synthesis is a highly regulated, dynamic process that is known to be altered during atherogenesis. Endothelial injury, which may be the primary event in atherosclerosis, has been reported to stimulate PG synthesis and accumulation in the arterial extracellular matrix. The objective of this investigation was to study injury-induced alterations in PG synthesis and accumulation in the neointima, developed in response to a selective balloon catheter de-endothelialization of aortas of normocholesterolaemic rabbits. One group of rabbit aortas was incubated with 35S-Na2SO4 for 8 h, to study in vitro the de novo synthesis of sulphated PG. Another group of rabbit aortas was used to study PG accumulated in aortic neointima vs. PG present in intima-media of normal aortas. Newly synthesized sulphated PG was characterized by light microscopic radioautography and size exclusion chromatography. Purified intimal-medial PG extracts from unlabelled aortas were analysed for protein and glycosaminoglycan (GAG) content and GAG distribution pattern. Results from this study revealed that the neointima of injured aortas synthesized sulphated PG at a significantly higher concentration than the intima of normal aortas. Size exclusion chromatography revealed that neointima synthesized higher molecular weight PG, and in a higher proportion, than its counterpart PG from normal aortas. PG accumulated in neointima of injured aortas showed a significantly altered GAG distribution pattern. These data confirm that neointima developed in response to injury synthesizes and accumulates PG which is altered compared to PG present in the intima-media of normal aorta.

Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs.

The present study was undertaken to investigate the effect of insulin-like growth factor-1 on proteoglycan synthesis and the autocrine/paracrine mechanism involving insulin-like growth factor-1 in the bovine coccygeal intervertebral disc. Insulin-like growth factor-1 stimulated proteoglycan synthesis in cultured cells of the nucleus pulposus of bovine intervertebral discs in a dose-dependent manner, and the effect was inhibited by an anti-insulin-like growth factor-1 monoclonal antibody. In situ hybridization histochemistry revealed the expression of insulin-like growth factor-1 mRNA in the cultured cells, and its production in these cells was demonstrated by radioimmunoassay. Insulin-like growth factor-1 receptor in the cultured cells was also demonstrated immunohistochemically. Scatchard analysis using an [125I]insulin-like growth factor-1 binding assay showed that the cells cultured in monolayer had a single type of insulin-like growth factor-1 receptor, whose affinity and number were estimated to be 7.38 x 10(8)/M and 9.27 x 10(4)/cell, respectively. These results suggest that insulin-like growth factor-1 stimulates proteoglycan synthesis in cells of the nucleus pulposus and that these cells in culture have an insulin-like growth factor-1 autocrine/paracrine mechanism. The expressions of insulin-like growth factor-1 mRNA and insulin-like growth factor-1 receptor in disc tissue were greater in cells of the nucleus pulposus of fetal bovine intervertebral discs than in those of the adult discs. These findings suggest that the action of autocrine/paracrine insulin-like growth factor-1 is more active in cells of the young nucleus pulposus than in cells of mature subjects.

Differentiation and Cell Surface Expression of Transforming Growth Factor-β Receptors Are Regulated by Interaction with Matrix Collagen in Murine Osteoblastic Cells

Although transforming growth factor (TGF)-beta enhances bone formation, it inhibits the differentiation of osteoblasts. To clarify the regulatory mechanism of osteoblastic differentiation and TGF-beta actions, the relationship among differentiation, TGF-beta actions, and matrix protein synthesis was examined using murine osteoblast-like MC3T3-E1 cells. Alkaline phosphatase (ALP) activity continued to increase during long-term cultures, and the increase was closely associated with a reduction in cell surface TGF-beta receptors competent to bind TGF-beta. Both the stimulation of proteoglycan synthesis and the inhibition of ALP activity by TGF-beta were also suppressed. Collagen synthesis inhibitors and an anti-alpha2beta1 integrin blocking antibody blocked the changes in ALP activity and TGF-beta receptors, and a DGEA peptide that interferes binding of collagen to alpha2beta1 integrin also blocked the increase in ALP activity. Furthermore, when MC3T3-E1 cells were cultured on extracellular matrix layers obtained from these cells, all the differentiation-associated changes could be observed without collagen production, and the extracellular matrix-induced differentiation was also blocked by an anti-alpha2beta1 integrin antibody. These results demonstrate that the interaction of cell surface alpha2beta1 integrin with matrix collagen synthesized by osteoblasts themselves is involved in the osteoblastic differentiation and the reduction in cell surface receptors and actions of TGF-beta. It is suggested that matrix collagen synthesized under the stimulation by TGF-beta plays an important role in the regulation of osteoblastic differentiation and TGF-beta actions by differentiation-associated down-regulation of TGF-beta receptors.

Effects of cyclic adenosine 3',5'-monophosphate on chondrocyte terminal differentiation and cartilage-matrix calcification.

We examined the effects of cyclic AMP on terminal differentiation and calcification in rabbit growth plate chondrocyte cultures. Dibutyryl cAMP (dbcAMP), as well as 8-bromo-cAMP abolished the increases in chondrocyte size, alkaline phosphatase activity, type X collagen synthesis, 1 alpha, 25-dihydroxyvitamin D3 receptor synthesis, the incorporation of 45Ca into insoluble material, and the calcium content. All of these occurred in parallel untreated cultures during the hypertrophic (terminal) stage. The inhibition of alkaline phosphatase by dbcAMP was detectable after 24 h, and this effect was reversible. dbcAMP and 8-bromo-cyclic AMP inhibited alkaline phosphatase induction and calcification at low concentrations (3-5 microM), whereas 10-30-fold higher concentrations were required to stimulate proteoglycan synthesis. These findings suggest that cAMP plays a crucial role in suppressing terminal differentiation of chondrocyte and cartilage-matrix calcification.

Changes in proteoglycan synthesis of chondrocytes in articular cartilage are associated with the time-dependent changes in their mechanical environment

Explant loading experiments were conducted to investigate the effect of load duration on proteoglycan synthesis. A compressive load of 0.1 MPa applied for 10 min was found to stimulate proteoglycan synthesis, while the same load applied for 20 h suppressed synthesis. This bimodal response suggests that the cells are responding to different mechanical stimuli as time progresses. A theoretical model has therefore been developed to describe the mechanical environment perceived by cells within soft hydrated tissues (e.g. articular cartilage) while the tissue is being loaded. The cells are modeled, using the biphasic theory, as fluid-solid inclusions embedded in and attached to a biphasic extracellular matrix of distinct material properties. A method of solution is developed which is valid for any axisymmetric loading configuration, provided that the cell radius, a, is small relative to the tissue height, h (i.e. h a ⪢ 1 ). A closed-form analytical solution for this inclusion problem is then presented for the confined compression configuration. Results from this model show that the mechanical environment in and around the cells is time dependent and inhomogeneous, and can be significantly influenced by differences in properties between the cell and the extracellular matrix.

Role of interleukin-1, tumor necrosis factor alpha, and interleukin-6 in cartilage proteoglycan metabolism and destruction. Effect of in situ blocking in murine antigen- and zymosan-induced arthritis.

To determine the involvement of interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6 in the cartilage pathology of murine antigen-induced arthritis (AIA) and zymosan-induced arthritis (ZIA). Arthritis was induced by intraarticular injection of zymosan in naive mice or by subcutaneous injection of methylated bovine serum albumin in sensitized animals. Mini-osmotic pumps releasing human recombinant IL-1 receptor antagonist (IL-1ra) protein were implanted intraperitoneally 2 days before arthritis induction, and neutralizing antibodies directed against murine IL-1 alpha, IL-1 beta, TNF alpha, or IL-6 were administered 1 day before. Proteoglycan (PG) synthesis and degradation were assessed in patellar cartilage. Murine IL-1 alpha and IL-1 beta injected intraarticularly at doses of 0.1-100 ng suppressed chondrocyte PG synthesis. The highest dose of TNF tested (100 ng) decreased PG synthesis marginally. In contrast, the maximum dose of IL-6 (1 microgram) stimulated PG synthesis 2 days after injection. Treatment of AIA with neutralizing monoclonal antibodies against either TNF alpha or IL-6 did not reduce either the PG degradation or the suppression of its synthesis. However, treatment with anti-IL-1 (alpha + beta) polyclonal antibodies totally prevented PG suppression, although the initial breakdown of PG was unaffected. This effect was confirmed when IL-1ra was administered in high doses. Moreover, treatment of ZIA with anti-IL-1 (alpha + beta), but not with anti-TNF, resulted in normal PG synthesis, confirming the key role played by IL-1 in the inhibition of PG synthesis. Treatment of AIA with anti-IL-1 did not affect inflammation during the acute phase, but a significant reduction of ongoing inflammation was noted at day 7, and there was a marked reduction in the loss of cartilage PG. The suppression of PG synthesis in both ZIA and AIA in mice is due to the combined local action of IL-1 (alpha + beta), and neither IL-6 nor TNF is involved. Moreover, the normalization of PG synthesis brought about by blocking of IL-1 ameliorates the cartilage damage associated with AIA.

Proteoglycan depletion alone is not sufficient to stimulate proteoglycan synthesis in cultured bovine cartilage explants

Articular cartilage comprises a small number of cells embedded within a matrix primarily composed of collagen and proteoglycan (PG). The functional integrity of the tissue is highly dependent on the maintenance of matrix structure, which, in turn, is controlled by the chondrocytes. In normal tissue there is a slow but steady turnover of matrix components such that their levels remain constant. In certain diseased states the equilibrium is upset, resulting in a net loss of matrix components. The object of the present study was to artificially upset the synthetic/loss equilibrium by enzymic depletion and assess the ability of chondrocytes to respond by increasing PG synthesis. Cultured bovine articular cartilage explants were depleted using enzymes as follows; 10 U/ml Streptomyces hyaluronidase (induced a 30% loss of PG), 2000 U/ml testicular hyaluronidase (70% loss of PG) and 100 U/ml collagenase (35% loss of PG) and control (6% loss of PG). Collagenase also induced a 50% loss of collagen. Collagenase treatment induced a 50% stimulation of PG synthesis above control levels. Elevated synthesis levels were maintained for 9 days. Testicular hyaluronidase induced a brief elevation in PG synthesis on day 3 of culture. Streptomyces hyaluronidase treatment did not cause an alteration in the rate of PG synthesis above control levels. Histological examination indicated that collagenase-treated explants formed outgrowths consisting of rounded chondrocytes within a fine fibrous matrix which stained intensely with safranin-O, indicating a high concentration of PG. The production of repair-like outgrowths may explain the elevated PG synthesis rates measured. It appears, therefore, that collagen and matrix organization is more important than PG levels in the control of PG synthesis in articular cartilage explant cultures.

The recovery of articular cartilage in explant culture from interleukin-1α: Effects on proteoglycan synthesis and degradation

The cytokine interleukin-1 (IL-1) strongly inhibits proteoglycan biosynthesis and increases extracellular proteoglycan degradation in cartilage. In this study we investigated how quickly proteoglycan turnover recovered after IL-1 treatment. Porcine articular cartilage in explant culture was incubated with recombinant human (rh) IL-1 alpha, rh insulin-like growth factor (IGF)-1 or rh-transforming growth factor (TGF)-beta 1 for 3 days. RhIL-1 alpha strongly inhibited synthesis and promoted matrix degradation over 3 days, whereas rhIGF-1 stimulated proteoglycan synthesis, and rhTGF-beta 1 had no significant effect on synthesis. In the absence of serum, the recovery of proteoglycan synthesis after IL-1 treatment (10 ng/ml) for 3 days was extremely slow, and it remained 30% inhibited even after a further 8 days of recovery. Matrix degradation of proteoglycan assessed from the rate of release of glycosaminoglycan into the culture medium recovered more quickly and was greatly reduced within 3 days. Shorter exposure of explants to IL-1 alpha (8 h) resulted in less inhibition of synthesis, but it did not result in a more rapid rate of recovery following its removal. Treatment of cartilage with IGF-1 (20 ng/ml) or TGF-beta (10 ng/ml) during the recovery period increased the rate of recovery of both synthesis and degradation. Synthesis recovered to control rates within 6 days and degradation within 2 days. TGF-beta (10 ng/ml) was slightly more potent than IGF-1. Fetal calf serum (10% v/v) also promoted recovery in a similar way to the growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucosamine sulfate compared to ibuprofen in osteoarthritis of the knee

Glucosamine sulfate is able to stimulate proteoglycan synthesis by chondrocytes and has mild anti-inflammatory properties. In clinical trials, glucosamine sulfate was more effective than placebo in controlling the symptoms of osteoarthritis (OA). In order to better characterize this therapeutic activity, we conducted a randomized, double-blind, parallel-group study of glucosamine sulfate 500 mg t.i.d. vs ibuprofen 400 mg t.i.d., orally for 4 weeks. The study included 200 hospitalized patients with active OA of the knee, symptoms for at least 3 months and a Lequesne's index of at least 7 points. Patients were evaluated weekly. Response was defined as a reduction in the Lequesne's index by at least 2 points if the enrollment value was higher than 12 points, or by at least 1 point if the enrollment value was 12 or less points, together with a positive overall assessment by the investigator. The improvement tended to be sooner under ibuprofen (48% responders vs 28% after the 1st treatment week; P = 0.06, Fisher's Exact test), but there was no difference from the 2nd week onward, with a success rate of 52% in the ibuprofen group and of 48% in the glucosamine group (P = 0.67) at the end of treatment. The average Lequesne's index at enrollment was around 16 points and decreased by over 6 points in both groups, again with the above described trend. On the other hand, 35% of patients on ibuprofen reported adverse events, mainly of gastrointestinal origin, vs 6% adverse events with glucosamine (P < 0.001, Fisher's Exact test). The number of adverse event related drop-outs was different between the two groups (7% vs 1%, respectively; P = 0.035). Glucosamine sulfate was therefore as effective as ibuprofen on symptoms of knee OA. These data confirm glucosamine sulfate as a safe symptomatic Slow Acting Drug for OA.

Antisense S-oligonucleotide against transforming growth factor-beta 1 inhibits proteoglycan synthesis in arterial wall.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent stimulator of proteoglycan (PG) synthesis by cultured smooth muscle cells. To test the hypothesis that TGF-beta 1 stimulates PG synthesis in whole artery wall we have investigated the effect of blocking endogenous TGF-beta 1 using an antisense S-oligonucleotide (ASO) directed against the first 7 codons for the N-terminal region of active TGF-beta 1. This sequence reduced TGF-beta 1 secretion by cultured endothelial cells by 40-55%. To determine the effect of the ASO on PG synthesis in whole vessel we chose the rat carotid artery (RCA) maintained in organ culture, a model in which we previously documented endothelial-dependent increases in PG synthesis over time in culture. We report here that the increases in PG in the inner layers of the vessel wall are matched by similar increases in TGF-beta 1 mRNA. To test for the effect of ASO on PG synthesis, small segments of RCA, maintained in organ culture (Medium 199, supplemented with 1% FCS), were exposed on day 6 to either control media, antisense TGF-beta 1, sense TGF-beta 1, or a non-sense sequence at 5, 10 and 20 microM concentrations. Following 24 h exposure to the oligonucleotides, cultures were labelled for a further 24 h with [3H]glucosamine and processed for autoradiography. Antisense TGF-beta 1 at 10 and 20 microM produced a 60% reduction in PG synthesis in the endothelium and adjacent first layer of the media.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of the synthesis of large proteoglycans in anatomically intact murine articular cartilage by steroids and insulin-like growth factor I.

The exact regulation of the synthesis of cartilage specific molecules, such as collagen type II and aggrecan, by articular chondrocytes is unknown, but growth factors and hormones probably play an important part. The effects of glucocorticosteroids (prednisolone and triamcinolone), in combination with insulin-like growth factor I (IGF-I), on the synthesis and hydrodynamic volume of proteoglycans from murine patellar cartilage were investigated. The in vitro effect of IGF-I and steroids on proteoglycan synthesis in murine patellar cartilage was evaluated by [35S]sulphate incorporation in combination with dissociative gel chromatography using a Sephacryl S-1000 column. The impact of in vivo prednisolone (0-5 mg/kg) on proteoglycan synthesis in murine patellar cartilage was analysed by [35S]sulphate incorporation immediately after dissection from the knee joint. Prednisolone stimulated proteoglycan synthesis in murine patellar cartilage from normal knees and in cartilage from knees injected with papain in vitro in the absence and presence of IGF-I. Moreover, oral administration of prednisolone for seven days to C57Bl10 mice resulted in enhanced proteoglycan synthesis in patellar cartilage. The incubation of patellar cartilage for 48 hours without serum or growth factors led to the synthesis of proteoglycans with a smaller hydrodynamic volume than those synthesised immediately after dissection of the patellae. This could either be circumvented by the addition of IGF-I or by the addition of glucocorticosteroids (prednisolone or triamcinolone) to the culture medium. These results show that in a dose range of 0.0003-0.3 mmol/l, glucocorticosteroids, like IGF-I, stimulate proteoglycan synthesis and maintain the synthesis of hydrodynamically large proteoglycans by chondrocytes from murine articular cartilage. This indicates that glucocorticosteroids might play a part in the preservation of matrix integrity in articular cartilage.

S5.16 Stimulation of proteoglycan synthesis by interleukin-4

S5.16 Stimulation of proteoglycan synthesis by interleukin-4

Role of proteoglycans and cytoskeleton in the effects of TGF-β1 on renal proximal tubule cells

Transforming growth factor-beta (TGF-beta) is a critical cell regulatory protein which influences cell growth, cell differentiation and cell chemotaxis. TGF-beta 1 has been previously shown to promote a migratory and adherent transformation of monolayers of renal proximal tubule cells in primary culture to form solid clusters of cells. To better understand the cellular basis of this TGF-beta 1 effect, these studies evaluated the influence of TGF-beta 1 on the synthesis of proteoglycans and on cytoskeleton rearrangement in rabbit renal proximal tubule cells in primary culture, and their role in this transformation effect of TGF-beta 1. Biosynthetic labeling of proteoglycans with 35S sulfate and enzyme digestion studies demonstrated that TGF-beta 1 promoted the synthesis of heparan sulfate proteoglycans in these cells. The importance of proteoglycan synthesis induced by TGF-beta 1 in this migration and aggregation process was demonstrated with the use of two chemically-dissimilar proteoglycan synthesis inhibitors: xyloside and galactosamine. Both compounds inhibited TGF-beta 1 stimulation of proteoglycan synthesis and diminished TGF-beta 1 promoted transformation of proximal tubule cells as assessed by quantitative morphometry. Further experiments evaluated the influence of TGF-beta 1 on actin microfilaments with the use of rhodamine conjugated phalloidin staining and immunofluorescent microscopy, and demonstrated that TGF-beta 1 provoked a dramatic rearrangement of actin microfilaments into stress fibers. The use of actin microfilament disrupting agents, cytochalasin B and D, attenuated the stress fiber formation promoted by TGF-beta 1 and inhibited the TGF-beta 1-induced morphologic transformation of these cells. Further studies evaluated these effects on the rate of DNA synthesis in these cells, as assessed with 3H-thymidine incorporation. Proteoglycan synthesis inhibitors significantly diminished the maximal proliferative response of these epithelial cells to epidermal growth factor (EGF). In contrast, actin microfilament disaggregation with cytochalasin B or D did not change the rate of DNA synthesis in response to EGF but did attenuate the antiproliferative effect of TGF-beta 1 on EGF-induced DNA synthesis cells. These studies demonstrate that the TGF-beta 1 promoted synthesis cells. These studies demonstrate that the TGF-beta 1 promoted an increase in the production of proteoglycans and a higher ordered structure of the cytoskeleton. Both effects were instrumental in the adhesive migratory response of proximal tubule cells to TGF-beta 1 as well as the DNA synthesis rate response to both EGF and TGF-beta 1.

Immunoglobulin G of patients with circumscribed pretibial myxedema of Graves' disease stimulates proteoglycan synthesis in human skin fibroblasts in culture.

Hyaluronic acid and proteoglycan accumulate in the affected skin of Graves' disease patients with pretibial myxedema (PTM). We aimed to determine whether an autoantibody IgG circulating in PTM patients stimulates proteoglycan synthesis in human skin fibroblasts, resulting in PTM. IgGs were purified from 14 normal subjects, 11 Graves' disease patients with PTM. 5 Graves' disease patients with active ophthalmopathy and 15 Graves' disease patients with neither PTM nor ophthalmopathy. Human skin fibroblasts were incubated with the IgGs and labeled with [35S]sulfate. The medium and cell layer were separated and the proteoglycan was extracted. The 35S radioactivity in the proteoglycan fraction was measured. Compared with normal IgGs or with those of Graves' disease without PTM or ophthalmopathy, PTM IgGs significantly increased the incorporation of the 35S into the proteoglycan. The effect of PTM IgG was dose-dependent. As PTM IgG did not alter degradation of the 35S labeled proteoglycan, an increase in 35S incorporation reflects increased synthesis. The effect was mediated through a mechanism other than adenylate cyclase activation. The present study demonstrates the presence of an autoantibody in PTM IgG that stimulates proteoglycan production through human skin fibroblasts. This is not correlated with the thyroid stimulating antibody activity. It is suggested that the activity of this antibody leads to the development of PTM.

Heparin stimulates proteoglycan synthesis by vascular smooth muscle cells while supressing cellular proliferation

We studied the effect of heparin on proteoglycan synthesis by bovine aortic smooth muscle cells in culture. Confluent, growth-arrested cells were incubated with [ 35S]sulfate, [ 3H]glucosamine or [ 3serine in the presence of 0–600 μg/ml heparin. Metabolically labeled proteoglycans secreted into the culture medium and associated with the cell layer were analyzed. In cultures treated with heparin there was a dose-dependent increase in [ 35S]sulfate incorporation into secreted proteoglycans which reached a maximum (35% above controls) at 100 μg/ml heparin. At higher concentrations of heparin, the stimulatory activity declined and finally disappeared. Radioactivity in cell-associated proteoglycans increased significantly (16% above controls) only in cultures treated with 100 μg/ml heparin. Heparin also produced similar increases in the incorporation of [ 3H]glucosamine and [ 3H]serine into secreted and cell-associated proteoglycans. While chondroitin sulfate, dermatan sulfate and heparan sulfate were elevated in the media, only chondroitin sulfate and heparan sulfate were increased in the cell layer. Heparin did not alter the degradation of proteoglycans. Heparin, while inhibiting the proliferation of subconfluent smooth muscle cells, also stimulated to a greater extent the incorporation of [ 35S]sulfate into proteoglycans. Other glycosaminoglycans, such as heparan sulfate, dermatan sulfate, heparin hexasaccharide and Sulodexide caused a significant but lesser stimulation of proteoglycan synthesis, while chondroitin sulfates and hyaluronic acid had no effect. Gel filtration chromatography of proteoglycans and their constituent glycosaminoglycans from heparin-treated and untreated cultures showed no differences in their molecular size. The results indicate that heparin can stimulate proteoglycan synthesis by vascular smooth muscle cells irrespective of their state of proliferation. This might have implications in vessel wall repair and arterial wall lipid deposition.

Demonstration of receptors for epidermal growth factor on cultured rabbit chondrocytes and regulation of their expression by various growth and differentiation factors

Epidermal growth factor (EGF) receptors were demonstrated on cultured rabbit costal chondrocytes. After crosslinking, the receptors on the cells with 125I-EGF, one major band of 170 KDa was separated by SDS-PAGE. Scatchard analysis demonstrated two classes of EGF receptors with Kd values of 0.3 nM and 1.6 nM. The numbers of high and low affinity receptors were 3,000 and 10,000 per cell, respectively. EGF receptors on chondrocytes were increased by treatment with retinoic acid and interleukin-1β, which inhibited proteoglycan synthesis. On the other hand, parathyroid hormone and dibutyryl cyclic AMP, which stimulated proteoglycan synthesis, decreased the number of EGF receptors. Treatments with these agents did not change the affinity of the receptors. These findings suggest that the number of EGF receptors is a negative marker of chondrocyte differentiation.

Effects of free and bound insulin-like growth factors on proteoglycan metabolism in articular cartilage explants.

This article describes the effects of bound forms of insulin-like growth factors (IGFs) on proteoglycan metabolism by bovine articular cartilage in explant culture. When these growth factors were added to articular cartilage explants complexed with their native serum binding proteins (BPs), both IGF-I-BP complex and IGF-II-BP complex stimulated proteoglycan synthesis to different degrees over a 3-day period. When added to the medium of cultures of articular cartilage over 5 days, IGF-II-BP complex induced high rates of synthesis and low rates of catabolism of proteoglycans, giving rise to tissue levels of proteoglycan similar to those observed in fresh tissue. When articular cartilage was maintained in culture with the same concentration of IGF-I-BP complex, tissue levels of proteoglycans fell over the culture period because of lower rates of proteoglycan synthesis. Analysis of the proteoglycans synthesized by articular cartilage in the presence of free or bound IGF-I or IGF-II showed that these growth factors stimulated the rate of synthesis of the large proteoglycan species present in cartilage but did not affect the synthesis of the small proteoglycans.

Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell-conditioned medium

We have previously shown (Berrou et al., J. Cell. Phys., 137:430-438, 1988) that porcine endothelial cell-conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. 1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]-sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. 2) Whereas ECCM stimulation of [35S]-methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine-labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. 3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine-labeled proteoglycans 7 protein cores of high apparent molecular mass (550-200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]-serine incorporation in response to ECCM (51%, as measured by Sepharose CL-6B chromatography). 4) Finally, incorporation of [35S]-sulfate into chondroitinase-generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL-6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that 1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and 2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.