Prostaglandin A2 and 35S uptake in connective tissue

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartlidge, long bone and skull bone than did normal control animals. Administration of prostaglandin A₂ stimulated ³⁵S uptake by lung, kidney and aorta while ³⁵S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.     

Prostaglandin A2 and 35S uptake in connective tissue

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartilage, long bone and skull bone than did normal control animals. Administration of prostaglandin A2 stimulated 35S uptake by lung, kidney and aorta while 35S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Cartilage-derived factor (CDF) I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture

A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of ³⁵SO₄^?2, [³H]-glucosamine and [³H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.     

Phagocytic release of human leukocyte neutral proteases: Analysis of cartilage degradation products

Human polymorphonuclear leukocyte neutral proteases (HLNP) released during the process of phagocytosis of aggregated human gamma globulin were tested for their ability to degrade intact rabbit ear cartilage. Using ³⁵S-labeled cartilage as substrate, HLNP derived from 45 × 10⁷ cells released about 45% of the total incorporated ³⁵S. DE-52 chromatography of incubation supernatants revealed a single ³⁵S peak associated with minimal quantities of peptide or protein material as estimated by absorbance at OD_{230 + 280 nm}. Analytical ultracentrifugation gave a molecular weight of 51,800. Incubation of cartilage with excess α-chymotrypsin released ³⁵S-containing protein and peptide elements (M_r 79,400). Therefore, degradation, of the proteoglycans of intact cartilage by HLNP is more extensive than that noted with bovine pancreas α-chymotrypsin. The products of HLNP and α-chymotrypsin digestion of cartilage contained chondroitin sulfates A and/or C since both materials (after column chromatography) were sensitive to chrondroitinase ABC and AC digestion. Collagenolytic activity of HLNP is minimal compared to proteolytic activity as evidenced by the lack of hydroxyproline containing peptides released from cartilage during enzyme incubation. It is suggested that HNLP incubated with intact cartilage may serve as a relevant model in the search for new agents which could combat enzyme-mediated cartilage destruction.     

The N-Terminal Cleavage of Chondromodulin-I in Growth-Plate Cartilage at the Hypertrophic and Calcified Zones during Bone Development

Chondromodulin-I (ChM-I) is a 20–25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp³⁷-Asp³⁸. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20–25 kDa form of ChM-I (ID₅₀ = 8 nM). Immunohistochemistry suggested that 20–25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20–25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20–25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.     

Turnover of proteoglycans in guinea pig costal cartilage

The turnover in vivo of proteoglycans of guinea pig costal cartilage was investigated using Na₂³⁵SO₄ as precursor. Proteoglycans were extracted with guanidine · HCl, at both low and high ionic strength, and purified and fractionated by ultracentrifugation in CsCl gradients under associative and dissociative conditions. The results suggest that the sulfate is incorporated into macromolecules of at least two major metabolic pools with half-lives of about 3 days and about 60–70 days, respectively. Molecules with a fast turnover were enriched in the low ionic strength extracts and in fractions containing small, nonaggregated proteoglycans. No substantial evidence was found for a precursor-product relationship between different fractions.     

The effect of ascorbic acid oxidation on the incorporation of sulfate by slices of calf costal cartilage

A marked inhibition of the incorporation of S³⁵-sulfate by normal calf costal cartilage was produced by potassium ascorbate in the presence of catalytic amounts of cupric ions. The effect of the various components of the ascorbic acid oxidizing system (potassium ascorbate, cupric ions, cuprous ions, hydrogen peroxide, dehydroascorbic acid) was investigated. The results of experiments in which hydrogen peroxide, catalase, or sodium azide were used singly or in combination suggest that the inhibition produced by the ascorbic acid oxidizing system is due, to a considerable extent, to the production of hydrogen peroxide. Dehydroascorbic acid was also found to inhibit the incorporation of S³⁵-sulfate by cartilage slices. However, the gradual fall in pH which resulted from the addition of dehydroascorbic acid could account to a large extent for the inhibitory effect observed because the incorporation of S³⁵-sulfate by cartilage slices decreases sharply as the pH is lowered. The incorporation of S³⁵-sulfate by cartilage slices is inhibited also by increasing the concentration of phosphate.     

Effect of retinoic acid on chondrocyte glycosaminoglycan biosynthesis.

The effect of retinoic acid on glycosaminoglycan biosynthesis was investigated in rat costal cartilage chondrocytes in vitro. At levels of 10^?9 to 10^?8m retinoic acid, ³⁵SO₄ uptake into glycosaminoglycans was reduced 50%. At these low levels of retinoic acid there was no evidence of lysosomal enzyme release. The results are explained best in terms of modification of glycosaminoglycan synthesis, rather than accelerated degradation. Retinoic acid selectively modified the incorporation of ³⁵SO₄ or [¹⁴C]glucosamine into individual glycosaminoglycans fractions under the conditions studied. The relative incorporation of radiolabeled precursor into heparan sulfate (and/or) heparin increased three- to fourfold. The relative incorporation of radiolabeled precursor remained constant for chondroitin 6-sulfate, whereas incorporation into chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Under the conditions studied, retinoic acid did not appear to be cytotoxic and did exhibit selective control over glycosaminoglycan biosynthesis. It is suggested that the decreased incorporation of ³⁵SO₄ into glycosaminoglycans at hypervitaminosis A levels of retinol may be accounted for by the presence of low levels of retinoic acid, a naturally occurring metabolite.     

Deficiency of fibroblast activation protein alpha ameliorates cartilage destruction in inflammatory destructive arthritis

IntroductionInflammatory destructive arthritis, like rheumatoid arthritis (RA), is characterized by invasion of synovial fibroblasts (SF) into the articular cartilage and erosion of the underlying bone, leading to progressive joint destruction. Because fibroblast activation protein alpha (FAP) has been associated with cell migration and cell invasiveness, we studied the function of FAP in joint destruction in RA.MethodsExpression of FAP in synovial tissues and fibroblasts from patients with osteoarthritis (OA) and RA as well as from wild-type and arthritic mice was evaluated by immunohistochemistry, fluorescence microscopy and polymerase chain reaction (PCR). Fibroblast adhesion and migration capacity was assessed using cartilage attachment assays and wound-healing assays, respectively. For in vivo studies, FAP-deficient mice were crossed into the human tumor necrosis factor transgenic mice (hTNFtg), which develop a chronic inflammatory arthritis. Beside clinical assessment, inflammation, cartilage damage, and bone erosion were evaluated by histomorphometric analyses.ResultsRA synovial tissues demonstrated high expression of FAP whereas in OA samples only marginal expression was detectable. Consistently, a higher expression was detected in arthritis SF compared to non-arthritis OA SF in vitro. FAP-deficiency in hTNFtg mice led to less cartilage degradation despite unaltered inflammation and bone erosion. Accordingly, FAP^−/− hTNFtg SF demonstrated a lower cartilage adhesion capacity compared to hTNFtg SF in vitro.ConclusionsThese data point to a so far unknown role of FAP in the attachment of SF to cartilage, promoting proteoglycan loss and subsequently cartilage degradation in chronic inflammatory arthritis.     

A “coupled” subchondral bone-articular cartilage tissue culture system for the study of cartilage proteoglycan metabolism

Summary Current evidence suggests that interactions between the subchondral bone and the articular cartilage of mammalian diarthrodial joints may occur through the action of bone-associated peptide factors. However, there is no suitable organ culture model for studying these interactions. This study defines a long-term tissue culture system where the articular cartilage is coupled to the adjacent subchondral bone obtained from the proximal ends of bovine metacarpals. Autoradiography done over 3 mo., by utilizing [³⁵S]SO₄ incorporation into cartilage proteoglycan (PG) and a procedure for cutting non-decalcified bone, demonstrated similar numbers of silver grains over chondrocytes in all cartilage zones, including the bone-cartilage interface. Newly synthesized PG (NSPG) from the cartilage of the “coupled” system over a 3-wk period was primarily of large hydrodynamic size (Kav of 0.34). Comparable bovine articular and nasal cartilage slice systems, incubated for short periods of time, yielded similar and somewhat larger NSPG, respectively. Labeled chondroitin sulphate PG accumulating in the medium of primary chondrocyte monolayer cultures, derived from the cartilage of the coupled system at 0, 1, 2, and 3 wk, revealed two polydisperse subpopulations (Kav of 0.30 to 0.38 and 0.51 to 0.68). We conclude that this coupled bone-cartilage system is viable for prolonged periods, is suitable for studies on the metabolism of articular cartilage PGs, and seems to have some advantages over the cultured articular cartilage slice system.     

Evidence that endogenous cyclic AMP does not modulate serum sulfation factor action on embryonic chicken cartilage

The role of cartilage cyclic AMP as a mediator or modulator of serum sulfation factor (SSF) action on embryonic chicken cartilage was assessed. Media with concentrations of rat serum (7.5%) sufficient to maximally stimulate chondromucoprotein synthesis as measured by ³⁵SO₄ incorporation did not change cartilage cyclic AMP levels. Theophylline (2.5mM) doubled cyclic AMP in cartilage incubated in media but had no effect on ³⁵SO₄ incorporation. In media containing 5% rat serum, theophylline at 0.5, 1.5 and 2.5mM caused a similar and significant rise in tissue cyclic AMP but only 2.5mM inhibited SSF stimulated ³⁵SO₄ incorporation. The data indicate that cartilage cyclic AMP neither mediates nor modulates SSF action on cartilage chondromucoprotein synthesis.     

BONE FORMATION INDUCED IN MOUSE THIGH BY CULTURED HUMAN CELLS

Cultured FL human amnion cells injected intramuscularly into cortisone-conditioned mice proliferate to form discrete nodules which become surrounded by fibroblasts. Within 12 days, fibroblastic zones differentiate into cartilage which calcifies to form bone. Experiments were conducted to test the hypothesis that FL cells behave as an inductor of bone formation. In the electron microscope, FL cells were readily distinguished from surrounding fibroblasts. Transitional forms between the two cell types were not recognized. Stains for acid mucopolysaccharides emphasized the sharp boundary between metachromatic fibroblastic and cartilaginous zones and nonmetachromatic FL cells. ³⁵S was taken up preferentially by fibroblasts and chondrocytes and then deposited extracellularly in a manner suggesting active secretion of sulfated mucopolysaccharides. FL cells showed negligible ³⁵S utilization and secretion. FL cells, labeled in vitro with thymidine-³H, were injected and followed radioautographically, during bone formation. Nuclear label of injected FL cells did not appear in adjacent fibroblasts in quantities sufficient to indicate origin of the latter from FL cells. The minimal fibroblast nuclear labeling seen may represent reutilization of label from necrotic FL cells. It is suggested that FL cells injected into the mouse thigh induced cartilage and bone formation by host fibroblasts.     

Timed appearance of a calcium-binding protein containing gamma-carboxyglutamic acid in developing chick bone

Bone contains a small protein, rich in the vitamin K-dependent calcium-binding amino acid γ-carboxyglutamate (Gla). This protein, named osteocalcin, is extractable by neutral EDTA demineralization and contains over 80% of the total peptide Gla found in bone. Osteocalcin binds Ca²⁺ ions with moderate affinity (2 moles of Ca²⁺/6500 g of protein; K_d = 0.83 mM). Osteocalcin appears in embryonic chick bones (mandible, calvaria, tibiotarsus, and femur) coincident with the first histologically observable deposition of bone mineral at 8 to 12 days after fertilization. The quantity of this protein increases dramatically during development with characteristic onset and kinetics for each type of bone. In the long bone diaphysis (midshaft), the fraction of noncollagen protein represented by osteocalcin increases 100- to 200-fold between the 8th and 20th day. Relative to total bone protein, the increase is about 35-fold. Osteocalcin may play a role in the development of mineralized tissues and may be a characteristic product of cells differentiated with respect to bone and/or cartilage formation.     

Purification of bovine somatomedin.

A procedure for the purification of bovine somatomedin (SM⁴) is presented. The purification scheme utilizes ultrafiltration through membranes of nominal mol. wt. cutoffs, molecular sieve chromatography and finally iso-electric focusing. Two peaks of SM activity, measured by the $\text{in vitro}$ stimulation of ³⁵S-Na₂SO₄ and ³H-thymidine uptake by costal cartilage, were present after focusing; an acidic component having a pI of 6.0 – 6.7 and a basic component having a pI in the range of 7.8 – 8.3. The acidic component comprised 2% of the initial activity and was 120,000-fold purified: the basic component comprised 10% of the initial activity and was 350,000-fold purified relative to the starting material. These components are similar in molecular size and pI to SM-A and SM-C isolated from human plasma.     

Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect

Biosynthesis of the undersulfated proteoglycan found in brachymorphic mouse (bm/ bm) cartilage has been investigated. Similar amounts of cartilage proteoglycan core protein, as measured by radioimmune inhibition assay, and comparable activity levels of four of the glycosyltransferases requisite for synthesis of chondroitin sulfate chains were found in cartilage homogenates from neonatal bm/bm and normal mice, suggesting normal production of glycosylated core protein acceptor for sulfation. When incubated with ³⁵S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS), bm/bm cartilage extracts showed a higher than control level of sulfotransferase activity. In contrast, when synthesis was initiated from ATP and ³⁵SO₄^2?, mutant cartilage extracts showed lower incorporation of ³⁵SO₄^2? into endogenous chondroitin sulfate proteoglycan (19% of control level) and greatly reduced formation of PAPS (10% of control level). Results from coincubations of normal and mutant cartilage extracts exhibited intermediate levels of sulfate incorporation into PAPS and endogenous acceptors, suggesting the absence of an inhibitor for sulfate-activating enzymes or sulfotransferases. Degradation rates of ³⁵S]PAPS and of ³⁵S-labeled adenosine 5′-phosphosulfate (APS) were comparable in bm/bm and normal cartilage extracts. Specific assays for both ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) showed decreases in the former (50% of control) and the latter (10–15% of control) enzyme activities in bm/bm cartilage extracts. Both enzyme activities were reduced to intermediate levels in extracts of cartilage from heterozygous brachymorphic mice (ATP-sulfurylase, 80% of control; APS kinase, 40–70% of control). Furthermore, the moderate reduction in ATP sulfurylase activity in bm/bm cartilage extracts was accompanied by increased lability to freezing and thawing of the residual activity of this enzyme. These results indicate that under-sulfation of chondroitin sulfate proteoglycan in bm/bm cartilage is due to a defect in synthesis of the sulfate donor (PAPS), resulting from diminished activities of both ATP sulfurylase and APS kinase, although the reduced activity of the latter enzyme seems to be primarily responsible for the defect in PAPS synthesis.     

RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis

Introduction

The receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.

Methods

Chronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E₂ (PGE₂), then further stained with tartrate-resistant acid phosphatase.

Results

Subchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE₂-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.

Conclusions

Chondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.     

Modifications of connective tissue matrices by an enzyme extracted from cartilage

Summary The degradative properties of an enzyme extracted from bovine costal cartilage were studied histochemically, autoradiographically, and electronmicroscopically. Previous work had indicated that this enzyme catalyzes the degradation of proteinpolysaccharide, light fraction (PP-L) from bovine costal cartilage to a proteinpolysaccharide with a lower protein content than the original substrate. Attempts were therefore made to ascertain if this protease was species specific and if proteinpolysaccharides other than those present in cartilage were susceptible to enzyme digestion. To this end, the effect of the enzyme on human fetal cartilage and jaws and the epiphyseal plates of neonatal and postnatal rats was studied. Furthermore, ³⁵S-H₂SO₄ was injected into pregnant rats, the fetuses were removed and sections of them were digested with the enzyme and then autoradiographed. In the histochemical experiments Alcian Blue with MgCl₂ was used for the staining of tissue polyanions and Bromphenol Blue for the detection of free basic groups. Finally, the limbs of 20 day-old rats were utilized in electronmicroscopic studies.Within the limitations of the techniques utilized the results obtained elucidated the following characteristics of the action of the enzyme on the tissues: 1. the products resulting from the action of this protease are more soluble than the proteinpolysaccharides originally present in the tissues, 2. the extent to which the enzyme affects the tissue depends directly on the state of maturation of the tissue and, therefore, on the state of aggregation of the matrix, and 3. the enzyme is not species or tissue specific.Autoradiograms of sections incubated with the enzyme also indicate an enhanced solubility of proteinpolysaccharides.The effects of the enzyme on ultrathin sections were manifested in an increased affinity for phosphotungstic acid staining of the extrafibrillar matrix and of particles in the lacunae of degenerating chondrocytes. The latter showed a distribution similar to that of particles seen in semithin sections stained with Azure A.Supported by Grant DE-02110-03-04 of the National Institutes of Public Health, Bethesda, Md.     

JNK1 is not essential for TNF-mediated joint disease

Tumour necrosis factor (TNF) signalling molecules are considered as promising therapeutic targets of antirheumatic therapy. Among them, mitogen-activated protein kinases are thought to be of central importance. Herein, we investigate the role in vivo of TNF-α signalling through c-Jun N-terminal kinase (JNK)1 in destructive arthritis. Human TNF transgenic (hTNFtg) mice, which develop inflammatory arthritis, were intercrossed with JNK1-deficient (JNK1 ^-/-) mice. Animals (n = 35) of all four genotypes (wild-type, JNK1 ^-/-, hTNFtg, JNK1 ^-/-hTNFtg) were assessed for clinical and histological signs of arthritis. Clinical features of arthritis (swelling and decreased grip strength) developed equally in hTNFtg and JNK1 ^-/-hTNFtg mice. Histological analyses revealed no differences in the quantity of synovial inflammation and bone erosions or in the cellular composition of the synovial infiltrate. Bone destruction and osteoclast formation were observed to a similar degree in hTNFtg and JNK1 ^-/-hTNFtg animals. Moreover, cartilage damage, as indicated by proteoglycan loss in the articular cartilage, was comparable in the two strains. Intact phosphorylation of JNK and c-Jun as well as expression of JNK2 in the synovial tissue of JNK1 ^-/-hTNFtg mice suggests that signalling through JNK2 may compensate for the deficiency in JNK1. Thus, JNK1 activation does not seem to be essential for TNF-mediated arthritis.     

Defective PAPS-synthesis in epiphyseal cartilage from brachymorphic mice

Activity levels of sulfotransferases, requisite for the sulfation of chondroitin sulfate proteoglycan, were measured in cell-free homogenates prepared from neonatal epiphyseal cartilage of normal C57B1/6J or homozygous brachymorphic mice. In the presence of [³⁵S]-PAPS only or [³⁵S]-PAPS plus an exogenous sulfate acceptor, comparable amounts of ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ were incorporated into chondroitin sulfate by the normal and mutant types of cartilage. In contrast, the mutant cartilage catalyzed the conversion of only 30% of the ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ into chondroitin sulfate as compared to normal mouse cartilage when synthesis was initiated from ATP and H₂³⁵SO₄. These results suggest that the production of an undersulfated proteoglycan which has previously been reported in brachymorphic mice (Orkin, R.W. $\text{et}\text{al}$. (1976) Devel. Biol. $\text{50}$, 82–94) may result from a defect in the synthesis of the sulfate donor PAPS.