Using genetic inference to re-evaluate the minimum longevity of the lemon shark Negaprion brevirostris.

A combination of mark-recapture and genetic sampling was used to extend the minimum longevity of an elasmobranch species and the life span estimate of the lemon shark Negaprion brevirostris was increased conservatively from 20·2 to 37 years. This increase in longevity means higher vulnerability and a longer recovery time from exploitation.

When bigger is not better: selection against large size, high condition and fast growth in juvenile lemon sharks.

Selection acting on large marine vertebrates may be qualitatively different from that acting on terrestrial or freshwater organisms, but logistical constraints have thus far precluded selection estimates for the former. We overcame these constraints by exhaustively sampling and repeatedly recapturing individuals in six cohorts of juvenile lemon sharks (450 age-0 and 255 age-1 fish) at an enclosed nursery site (Bimini, Bahamas). Data on individual size, condition factor, growth rate and inter-annual survival were used to test the 'bigger is better', 'fatter is better' and 'faster is better' hypotheses of life-history theory. For age-0 sharks, selection on all measured traits was weak, and generally acted against large size and high condition. For age-1 sharks, selection was much stronger, and consistently acted against large size and fast growth. These results suggest that selective pressures at Bimini may be constraining the evolution of large size and fast growth, an observation that fits well with the observed small size and low growth rate of juveniles at this site. Our results support those of some other recent studies in suggesting that bigger/fatter/faster is not always better, and may often be worse.

Expression of the tissue inhibitor of metalloproteinases (TIMP) gene family in normal and osteoarthritic joints.

The pathophysiological and biological significance of tissue inhibitor of the metalloproteinases-3 (TIMP-3) gene compared to other TIMPs was investigated in osteoarthritic (OA) human and normal bovine joint tissues. Human OA synovial fibroblasts in culture constitutively expressed TIMP mRNAs. TIMP-3, TIMP-1 and gelatinase A mRNAs were elevated in most human OA synovia over controls, while TIMP-2 expression was similar. TIMP-3 and TIMP-1 mRNAs present in bovine cartilage were inducible by serum factors. Transforming growth factor beta (TGF-beta 1) induced TIMP-3 RNA and protein in human OA and normal bovine chondrocytes. TIMP mRNAs were low (TIMP-1) or undetectable in human fetal chondrocytes but were expressed at all other ages. Thus, the two main joint tissues, synovial membranes and cartilage, express TIMP genes. Due to their matrix protecting activities, the presence of multiple TIMPs may be beneficial for normal joints, while increased TIMP-3 and TIMP-1 expression in arthritic joints may be associated with pathological remodeling.

Up-regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-beta in articular chondrocytes is mediated by serine/threonine and tyrosine kinases.

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn-over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF-beta), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP-3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF-beta-mediated induction of TIMP-3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased TIMP-3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP-3 gene to TGF-beta. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF-beta induction of TIMP-3. H7 and genistein also suppressed TGF-beta-induced TIMP-3 protein expression. These results suggest that TGF-beta signaling for TIMP-3 gene induction involves H7-sensitive serine/threonine kinase as well as herbimycin A- and genistein-sensitive protein tyrosine kinases.

The effects of tenidap on canine experimental osteoarthritis: II. Study of the expression of collagenase-1 and interleukin 1beta by in situ hybridization.

OBJECTIVE: To examine the effect of tenidap on the expression of collagenase- and interleukin 1beta (IL-1beta) genes in experimental canine osteoarthritis (OA). METHODS: The anterior cruciate ligaments of the right stifle joints of experimental dogs were sectioned by a stab wound incision and tissues samples were taken. Four dogs had received no treatment, 4 were treated with oral omeprazole (20 mg/day), and another 4 were treated with tenidap (3 mg/kg/bid) and omeprazole (20 mg/day). The dogs received medication for 8 weeks; all dogs were sacrificed at the end of this period. Tissues from 4 healthy dogs were used as controls. IL-1beta and collagenase-1 gene expression were measured in synovial membrane (synovial lining cells and mononuclear cell infiltrate) and collagenase-1 expression in cartilage using in situ hybridization techniques. Results were calculated as the percentage of cells expressing the gene, and expressed as cell score. RESULTS: The collagenase-1 cell score in the full thickness samples was significantly higher in OA cartilage than in normal cartilage, both in condyles and plateaus (p < 0.0002). Tenidap treated dogs showed a significantly lower cell score in femoral condyles and tibial plateaus in the superficial (p < 0.0002), deep (p < 0.005, p < 0.002, respectively), and full thickness (p < 0.0002) layers compared to OA dogs. No staining for collagenase-1 was observed in normal membrane. In OA synovial membrane, the collagenase-1 cell score was high in both the synovial lining cells and mononuclear cell infiltrate. Tenidap treated dogs showed a significantly lower score compared to OA tissue in both the synovial lining cells (p < 0.03) and the mononuclear cell infiltrate (p < 0.03). The relative decrease in the collagenase-1 cell score in the tenidap treated dogs was more pronounced in the mononuclear cell infiltrate. Staining for IL-1beta was observed in only a few lining cells in normal synovial membrane from unoperated dogs. In OA synovial membrane from untreated dogs, staining for IL-1beta was intense and was found in all dog specimens. The cell score was significantly higher in OA lining cells (p < 0.03) and mononuclear cell infiltrate (p < 0.03) compared to normal. In tenidap treated dogs, the score for IL-1beta was significantly lower than in OA, both in synovial lining cells (p < 0.03) and mononuclear cell infiltrate (p < 0.03). CONCLUSION: Tenidap significantly reduced in vivo expression of collagenase-1 and IL-1beta in experimental OA. These data are an extension of our previous study and showed that tenidap exerts its protective effects on OA lesions, likely by reducing the catabolic pathways of the disease.

Osteoarthritic synovial fibroblasts possess an increased level of tumor necrosis factor-receptor 55 (TNF-R55) that mediates biological activation by TNF-alpha.

OBJECTIVE: To investigate the presence, number, and level of expression of tumor necrosis factor receptors (TNF-R) in normal and osteoarthritic (OA) human synovial fibroblasts; to examine which receptor isotype mediates the biological response of these cells to TNF-alpha; and to study homologous regulatory mechanisms of TNF-R by TNF-alpha. METHODS: We used radioligand binding assay with [125I]TNF-alpha and flow cytometric analysis with specific antireceptor antibodies to characterize receptor populations, densities, and ligand induced internalization of TNF-R. Inducible cyclooxygenase (COX-2) synthesis, prostaglandin E2 (PGE2) release, and TNF-R shedding (soluble receptors, TNF-sR) were measured after incubation with TNF-alpha the presence or absence of receptor specific blocking antibodies. RESULTS: Although radioligand binding assays showed no difference in the density or affinity of TNF-R in OA synovial fibroblasts compared with normal cells, flow cytometric analysis revealed that OA cells express a significantly higher level of TNF-R55 (p < 0.04) than normal cells. The TNF-R55 was found to be the major receptor species responsible for ligand binding activity, such as COX-2 induction and PGE2 synthesis, since a specific antireceptor TNF-R55 blocking antibody inhibited about 76% of TNF-alpha binding and TNF-alpha stimulated COX-2 induction and PGE2 production. Further experiments revealed that TNF-R55 was the only receptor type internalized after binding TFN-alpha, whereas TNF-R75 was concomitantly shed. Moreover, reducing the shedding of TNF-sR, particularly the TNF-sR75, with a synthetic inhibitor decreased TNF-alpha induced PGE2 production. CONCLUSION: TNF-R55 is the major receptor isoform transducing PGE2 and COX-2 responses to TNF-alpha in OA synovial fibroblasts; soluble receptors could be involved in facilitating the binding of TNF-alpha to its receptor.

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: role of calcium and protein kinase A and C.

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E2 (PGE2) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE2 provoked a 3.9 +/- 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca++ channel blockers, verapamil and nifedipine, and the Ca++/calmodulin inhibitor, W-7. The Ca+2 ionophore, ionomycin, mimicked the effects of PGE2 as did the phorbol ester PMA, which activates Ca++/-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE2 did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE2 secretagogue, IL-1 beta, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 +/- 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE2 does not mediate the effect of its secretagogue and that IL-1 beta signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2 receptor signalling pathways. Taken together, our results suggest that PGE2 modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism.

Modulation of matrix metalloprotease 13 (collagenase 3) gene expression in equine chondrocytes by interleukin 1 and corticosteroids.

OBJECTIVE: To determine whether matrix metalloprotease 13 (MMP-13; collagenase 3) is produced by equine chondrocytes and to investigate modulation of its expression by recombinant human interleukin 1 beta (rhIL-1 beta) and corticosteroids. PROCEDURE: Equine chondrocytes in monolayer culture were stimulated with rhIL-1 beta. Total RNA was extracted, purified, and reverse transcribed into DNA. Using appropriate primers, a putative MMP-13 fragment was amplified by polymerase chain reaction, and cloned into a bacterial vector. The resultant fragment was purified and sequenced, then was used to prepare a digoxigenin-labeled cRNA probe. Monolayer cultures of first-passage chondrocytes were treated with rhIL-1 beta in the presence or absence of dexamethasone (10(-6)M) or methylprednisolone acetate (10(-9)M to 10(-5)M), in addition to positive and negative controls. Cellular RNA was extracted and resolved on agarose gels and subjected to northern blot analysis, using the equine MMP-13 probe. RESULTS: Reverse transcriptase-polymerase chain reaction enabled isolation of a 0.6-kb fragment of equine MMP-13 cDNA that had 93% homology with the human MMP-13 cDNA sequence. rhIL-1 significantly stimulated MMP-13 expression in the chondrocytes. Methylprednisolone acetate inhibited the stimulatory effects of rhIL-1 in dose-dependent manner that was statistically significant at 10(-5)M. CONCLUSIONS: Novel information was gained on the existence of MMP-13 and its expression in equine chondrocytes, which suggests a possible role for this enzyme in matrix degradation in horses with arthritis.

Asymmetric aldol condensation as a route to polypropionate derived pheromones.

The synthesis of the polypropionate-derived pheromones sitophilate (1) and sitophilure (2) are described using an asymmetric aldol condensation as the key step to adduct 6; compound 6 was smoothly converted to the antipodes of each pheromone. This procedure can be expanded to more complicated structures with the same type of syn configuration such as stegobinone (3) and serricornin (4).

Modulation of the Expression of Glucocorticoid Receptors in Synovial Fibroblasts and Chondrocytes by Prostaglandins and NSAIDs.

Endogenous glucocorticoids are of prime importance in the maintenance of cellular homeostasis through their binding to specific cell receptors. Under normal conditions, physiological concentrations of cortisol are potentially capable of suppressing metalloprotease synthesis by chondrocytes and synoviocytes. This hormone action is like to represent one of the major pathways through which the catabolism of cartilage under physiological conditions is kept in a homeostatic state. In osteoarthritis (OA), a severe reduction (about 50%) in the glucocorticoid receptor (GR) level in chondrocytes was found to be sufficient to alter the cellular steroid resistance in terms of the synthesis of destructive enzymes. Prostaglandins of the E series and dibutyryl cyclic AMP increased the GR level in normal and OA human articular chondrocytes. Synthetic PGE(1) (misoprostol) also induced upregulation of GR expression in chondrocytes. Naproxen and indomethacin, but not tiaprofenic acid, at therapeutic concentrations, significantly reduced the level of GR in synovial cells. This effect could be reversed by the additionl of misoprostol. These findings bring insight into the differential effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the GR system and may provide an explanation for the reduced level of GR found in OA chondrocytes. The addition of synthetic prostaglandins may prove to be of therapeutic importance in the treatment of arthritic diseases by potentiating the effects of therapeutically administered glucocorticoid on the reducation of the catabolism of articular cartilage.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA is constitutively expressed in bovine, human normal, and osteoarthritic articular chondrocytes.

Tissue inhibitors of metalloproteinases (TIMPs) inhibit the extracellular matrix (ECM) metalloproteinases (MMPs). To determine the source of TIMPs in synovial fluids of patients with osteoarthritis (OA), the ability of chondrocytes to express TIMP-2 and its regulation by agents found in inflammed joints was investigated. The constitutive TIMP-2 mRNA expression was demonstrated in chondrocytes from normal bovine, human OA and normal cartilage. The cross-hybridization of human and bovine TIMP-2 suggested its evolutionary conservation. Serum, IL-1, IL-6 and TGC-beta were unable to augment considerably the basal expression of TIMP-2 mRNA. TIMP-1 RNA expression in chondrocytes from human OA cartilage was elevated compared to non-OA chondrocytes, while TIMP-2 mRNA levels were similar in both. IL-1 beta, IL-6 and TGF-beta did not affect TIMP-2 expression but TGF-beta induced TIMP-1 mRNA in human OA chondrocytes. TIMP-2 and TIMP-1 are therefore differentially regulated in chondrocytes and the basal TIMP-2 levels may be needed for the cartilage ECM integrity.

The in vivo effects of intraarticular corticosteroid injections on cartilage lesions, stromelysin, interleukin-1, and oncogene protein synthesis in experimental osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is characterized by a progressive erosion of the articular cartilage. Studies suggest that cytokines and metalloproteases play an important role in this process. Previously, we found that corticosteroids given prophylactically reduce the severity of cartilage lesions. In this study, we examined and compared the in vivo efficacy of a corticosteroid, triamcinolone hexacetonide (TH), administered either prophylactically or therapeutically, on OA lesions and on cartilage metalloproteases, interleukin-1 beta (IL-1 beta), and oncogene synthesis in experimental OA. EXPERIMENTAL DESIGN: This study examines the effect of intraarticular injections of TH on the progression of lesions in the experimental dog model of OA. The animals had the anterior cruciate ligament of the right knee sectioned and were killed 12 weeks after surgery. A total of 30 dogs were used in four groups: group 1 (n = 8) had no treatment; group 2 (n = 8; TH-3) received TH at time of surgery and at 4 and 8 weeks later; group 3 (n = 7; TH-2) received TH at 4 and 8 weeks after surgery; and group 4 (n = 7; TH-1) received TH at 8 weeks after surgery. Knee cartilage was submitted to macroscopic and microscopic examinations, as well as immunohistochemical studies for stromelysin, IL-1 beta, and oncoproteins, c-Fos and c-Myc. RESULTS: Injections with TH significantly reduced the size of osteophytes in dogs from groups TH-3 (p < 0.0001) and TH-2 (p < 0.0002). The histologic severity of cartilage lesions was significantly reduced on condyles in all TH groups and on plateaus of dogs from groups TH-3 and TH-2. Immunohistochemistry revealed that TH significantly reduced the percentage of immunoreactive chondrocytes for stromelysin, and the effect was proportional to the number of injections received. For c-fos and c-myc, the reduction was particularly significant in the TH-3 and TH-2 groups. For IL-1 beta, the reduction was significant only in the TH-3 group. CONCLUSIONS: This study provides novel data showing the protective effect of corticosteroid injections on OA cartilage lesions not only under prophylactic but also under therapeutic conditions. The effect of TH may be mediated through a direct reduction in the expression and synthesis of proteolytic enzymes, such as stromelysin. Alternatively, the steroid could act by inhibiting the stimulation of protease synthesis by cytokines/oncogenes (IL-1 beta, c-Fos, and c-Myc).

Interleukin-1 beta induction of tissue inhibitor of metalloproteinase (TIMP-1) is functionally antagonized by prostaglandin E2 in human synovial fibroblasts.

Elevated levels of tissue inhibitor of metalloproteases-1 (TIMP-1) have been demonstrated in inflamed synovial membranes, and it is believed that the inhibitor may play a critical role in the regulation of connective tissue degradation. The present study was undertaken to define the cellular mechanism of action of the inflammatory mediators, interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE2), in the control of TIMP-1 synthesis and expression in human synovial fibroblasts. Recombinant human IL-1 beta induced a time- and dose-dependent saturable response in terms of TIMP-1 mRNA expression (effective concentration for 50% maximal response, EC50 = 31.5 +/- 3.3 pg/ml) and protein synthesis (EC50 = 30 +/- 3.3 pg/ml). The protein kinase C (PKC) inhibitors, H-7, staurosporine, and calphostin C, reversed the rhIL-1 beta induction of TIMP-1 mRNA. PGE2 also inhibited rhIL-1 beta-stimulated TIMP-1 mRNA expression and protein secretion in a dose-dependent fashion. The concentration of PGE2 necessary to block 50% of rhIL-1 beta-stimulated TIMP-1 secretion, IC50, was 1.93 ng/ml (4.89 nM). Forskolin, and other stable derivatives of cAMP, mimicked, to a large extent, the effects of PGE2. The phorbol ester, PMA, up-regulated considerably the mRNA expression of TIMP-1 but had no effect on protein production. Calphostin C substantially reduced PMA-activated TIMP-1 expression. Staurosporine, calphostin C, H-7, and substances that elevate cellular levels of cAMP, like PGE2, also reduced basal expression and synthesis of TIMP-1. Taken together, the data suggest that PKA and C may mediate opposing effects in terms of TIMP-1 expression and secretion in human synovial fibroblasts.

Increased insulin-like growth factor 1 production by human osteoarthritic chondrocytes is not dependent on growth hormone action.

OBJECTIVE: To investigate insulin-like growth factor 1 (IGF-1) production in normal and osteoarthritis (OA) chondrocytes and to further examine the role of growth hormone (GH) in adult human cartilage and, in particular, in diseased tissue. METHODS: IGF-1 production was measured with a radioimmunoassay. Binding assay, Northern blot, and reverse transcriptase polymerase chain reaction (RT-PCR) techniques were used for GH receptor (GHR) detection. The biological response to GH was estimated via IGF-1 production. RESULTS: We observed that basal levels of IGF-1 production were significantly higher in OA chondrocytes than in normal cells (P < 0.005). Adult human chondrocytes, however, were unresponsive to GH stimulation with regard to IGF-1 production, as shown in dose-response (0-1,000 ng/ml) and time-course (days 1-8) studies. In addition, no specific 125I-GH binding was detected in either cell type. Northern blot analysis revealed a 5.5-kb GHR messenger RNA (mRNA) species, but semiquantitative RT-PCR revealed no difference in GHR mRNA expression by normal and OA chondrocytes. CONCLUSION: This study indicates that the elevated synthesis of IGF-1 by adult human OA chondrocytes occurs through a GH/GHR-independent mechanism, suggesting that other factors are capable of controlling local IGF-1 production in these cells.

Coordinate regulation of matrix metalloproteases and tissue inhibitor of metalloproteinase expression in human synovial fibroblasts.

We examined the common signal transduction mechanisms governing collagenase (MMP-1), stromelysin-1 (MMP-3), and tissue inhibitor of metalloproteases (TIMP-1) gene expression in human synovial fibroblasts for insight into the pathophysiology of arthritis. MMP-1, MMP-3, and TIMP-1 expression and synthesis were induced in cultured human synoviocytes with recombinant human interleukin 1 beta in the absence or presence of either chemical inhibitors of protein kinase A and C (PKA, PKC), or prostaglandin E2, or cyclic AMP (cAMP) mimetics. We used enzyme immunoassays (EIA) to determine MMP-1, MMP-3, and TIMP-1 antigen levels in spent culture medium and Northern hybridization to measure steady state mRNA expression levels. Extracellular signals (e.g., IL-1, phorbol myristic acetate) that result in the activation of cytoplasmic PKC augment in tandem the expression and synthesis of MMP-1, MMP-3, and TIMP-1 in human synovial fibroblasts. In addition, such signals induce nuclear transcription factors (e.g., activator protein 1) that bind to common gene regulatory elements and augment promoter activity of MMP-1, MMP-3, and TIMP-1 gene promoter constructs. In contrast, signals that activate PKA oppose PKC mediated signals, in that the expression of MMP-1, MMP-3, and TIMP-1 are suppressed. Experimental data suggest that the expression of MMP-1, MMP-3, and TIMP-1 are coordinated through a series of common cytoplasmic signal transducing pathways, cis regulatory elements, and nuclear trans acting factors.

The reduced expression of glucocorticoid receptors in synovial cells induced by nonsteroidal antiinflammatory drugs can be reversed by prostaglandin E1 analog.

OBJECTIVE: We examined the effect of 3 commonly used nonsteroidal antiinflammatory drugs (NSAID), indomethacin, naproxen and tiaprofenic acid, and a prostaglandin E1 analog, misoprostol, on the glucocorticoid receptor level in synovial fibroblasts. METHODS: Synovial fibroblasts were isolated by enzymatic digestion from human normal synovial membranes. These cells were incubated with therapeutic and pharmacological concentrations of NSAID in the presence or absence of misoprostol (0.1-100 ng/ml). The glucocorticoid receptor (GR) level was measured by binding assay using 3H-dexamethasone. RESULTS: Naproxen and indomethacin but not tiaprofenic acid, at therapeutic concentrations, significantly reduced the level of GR in synovial cells. This effect was reversed with the addition of misoprostol. CONCLUSION: Our findings bring insight into the differential effects of NSAID on the GR system and may provide an explanation for the reduced level of GR found in OA chondrocytes. The possible interference with the action of therapeutically administered corticosteroids is discussed.

Prostaglandins E2 and E1 inhibit cytokine-induced metalloprotease expression in human synovial fibroblasts. Mediation by cyclic-AMP signalling pathway.

BACKGROUND: Cytokine modulated matrix metalloprotease (MMP, i.e., collagenase and stromelysin) synthesis may be associated etiologically with osteoarthritic diseases. The aim of this study was to investigate the mode of action by which interleukin-1 beta (IL-1 beta) induced collagenase and stromelysin mRNA expression and synthesis in normal human synoviocytes and to explore mechanisms of suppression of these proteolytic enzymes by prostaglandins (PG). EXPERIMENTAL DESIGN: Collagenase and stromelysin expression and synthesis were induced in cultured human synoviocytes with rhIL-1 beta in the absence or presence of either chemical inhibitors of protein kinase (PK) A and C, PGE2 or PGE1, or cAMP mimetics. We used enzyme immunoassays to determine MMP antigen levels in spent culture medium and Northern hybridization to measure steady-state MMP mRNA expression. RESULTS: Dose-response experiments revealed that 10 pg/ml of interleukin-1 beta was an effective sub-saturating concentration for the induction of collagenase and stromelysin expression in normal human synovial fibroblasts. The rate of collagenase synthesis and expression peaked at 18 to 24 hours after rhIL-1 beta stimulation, whereas stromelysin output increased steadily within the experimental time frame (72 hours). Protein kinase C inhibitors, H-7 and staurosporine, prevented the rhIL-1 beta induction of MMP mRNA expression and protein synthesis. Pretreatment of synoviocytes with phorbol myristate acetate for 18 hours abrogated the ability of rhIL-1 beta to induce MMP synthesis. Prostaglandins E2 and E1 potently inhibited in a dose-dependent fashion rhIL-1 beta induced MMP synthesis: PGE2, IC50, collagenase, 2.3 ng/ml; stromelysin, 21.2 ng/ml; PGE1, IC50, collagenase, 2.5 ng/ml; stromelysin, 13.4 ng/ml. MMP mRNA steady-state levels were suppressed in a fashion similar to that of the protein synthesis. Forskolin, dibutyryl cAMP, and 3-isobutyl-1-methyl xanthine mimicked the effects of the prostaglandins (PGs). [N-(2-methyl-amino)-5-isoquinoline-sulfonamide dihydrochloride] (H-8), an inhibitor of PKA activity, could reverse to a large extent, the suppressive effects of the PGs as did cycloheximide when preincubated with PGE2 before rhIL-1 beta activation of synoviocytes. CONCLUSIONS: We conclude that IL-1 beta stimulates MMP synthesis by activating PKC but not PKA, and that the synthesis of collagenase and stromelysin is discoordinate on a temporal and quantitative basis. PGs inhibit rhIL-1 beta-induced MMP expression and synthesis by virtue of their ability to increase cAMP intracellular levels and subsequent activation of signal transduction mechanisms involving PKA. Homeostasis may be maintained in acute episodes of joint inflammation through feedback processes involving locally produced eicosanoids.

Transcriptional regulation of plasminogen activator inhibitor-1 expression in human synovial fibroblasts by prostaglandin E2: mediation by protein kinase A and role of interleukin-1.

Differential expression of PAI-1 in connective tissues has been associated etiologically with some forms of arthritis. Our objective was to delineate the mechanisms by which PGE2 and IL-1 beta, inflammatory mediators commonly found at sites of inflammation, regulate the expression and synthesis of PAI-1 in human synoviocytes. PGE2 (and PGE1) inhibited PAI-1 mRNA expression and secretion in a dose-dependent manner with an IC50 (for antigen secretion) of 4.6 x 10(-10) M and 8.7 x 10(-10) M, respectively. Cyclic AMP agonists forskolin, Sp-cAMP, and IBMX mimic the effects of the PGEs. rhIL-1 beta stimulated the secretion of PAI-1 in a dose-dependent fashion under basal culture conditions; the effect was reversed by actinomycin D and the protein kinase inhibitors H7 and staurosporine but not KT-5720. PMA, an activator of protein kinase C, transiently increased (maximum 3 h) the expression of PAI-1 mRNA by approximately 10-fold, especially the 3.2 kb species. However, there was no significant increase in PAI-1 antigen secreted into the culture medium after PMA (100-300 nM) treatment. The half-life (t1/2) of PAI-1 mRNA, both the 3.2 and 2.2 transcripts was about 9.6 h (mean n = 3) and PGE2 has no affect on the stability of both messages. PGE2 reduced the rate of PAI-1 gene transcription as judged by run-off assays. The NSAID naproxen (30 micrograms/ml) induced the expression of PAI-1 mRNA over basal levels and super-induced the inhibitor's expression above rhIL-1 beta stimulated levels. Our results suggest that PGE2 suppresses PAI-1 expression and synthesis by activation of the cAMP/PKA system and inhibition of the rate of gene transcription. Data concerning the activation of PKC suggest that the expression, synthesis and release of the PAI-1 may be differentially regulated in normal human synoviocytes.

Human osteoarthritic chondrocytes possess an increased number of insulin-like growth factor 1 binding sites but are unresponsive to its stimulation. Possible role of IGF-1-binding proteins.

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: Binding experiments revealed a single class of binding sites. The mean +/- SEM affinity (Kd) of normal chondrocytes was 1.4 +/- 0.4 nM, with 26.8 +/- 5.5 x 10(3) binding sites/cell. OA chondrocytes had a lower affinity (Kd 15.4 +/- 4.7 nM) and a higher density (1,178.3 +/- 299.5 x 10(3) binding sites/cell) compared with normal cells (P < 0.004 and P < 0.001, respectively). Immunohistochemical studies with a monoclonal antibody (MAb) against the type 1 IGF receptor (alpha IR3) 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 alpha IR3 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.

Regulation of human normal and osteoarthritic chondrocyte interleukin-1 receptor by antirheumatic drugs.

OBJECTIVE: To determine the effect of antirheumatic drugs and corticosteroids on interleukin-1 receptor (IL-1R) levels in, and IL-1-stimulated metalloprotease synthesis and expression by, normal and osteoarthritic (OA) human articular chondrocytes. METHODS: IL-1R affinity and density of human chondrocytes were determined using radioligand binding experiments. Collagenase and stromelysin synthesis activities were analyzed by 14C-labeled type I collagen and Azocoll assays, respectively. Their messenger RNA (mRNA) levels were determined by Northern blot analysis. IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, and beta 2-microglobulin were measured using enzyme-linked immunosorbent assays. Protein synthesis was determined by 3H-leucine incorporation. RESULTS: Antirheumatic drugs reduced the IL-1R level in normal and OA chondrocytes in a dose-dependent manner. In normal chondrocytes, tenidap reduced the IL-1R level by 44% at 5 micrograms/ml to 88% at 100 micrograms/ml (50% inhibition constant [IC50] 10.1 micrograms/ml), indomethacin reduced IL-1R by 6% at 1.5 micrograms/ml to 43% at 60 micrograms/ml, and naproxen reduced IL-1R by 10% at 10 micrograms/ml to 41% at 300 micrograms/ml; the effects observed with indomethacin and naproxen occurred only when the drugs were used at levels above their therapeutic concentrations. In OA chondrocytes, the effect of indomethacin and naproxen on the IL-1R level was greatly reduced, whereas tenidap still had a marked effect (IC50 22.5 micrograms/ml). Dexamethasone and hydrocortisone had no consistent effect on the IL-1R level. At a therapeutic concentration (20 micrograms/ml), tenidap was found to reduce the IL-1R level in a time-dependent manner, with maximum inhibition (98%) by 48 hours. Tenidap was also found to markedly reduce collagenase and stromelysin synthesis and mRNA levels in IL-1-stimulated chondrocytes. CONCLUSION: The suppressive effects of tenidap on IL-1-stimulated metalloprotease synthesis and expression in OA and normal chondrocytes are likely related to a decrease in IL-1R levels. At therapeutic concentrations, tenidap has a greater effect on the IL-1R level than is seen with indomethacin or naproxen, and glucocorticoids have no effect on IL-1R.