John A. DiBattista

Are cytokines involved in osteoarthritic pathophysiology

The putative role and mechanism of action of cytokines in the progression of arthritic diseases such as osteoarthritis (OA) has received particular attention because of the important interaction between articular cartilage and synovium in the pathophysiology of the diseased state. Maintaining matrix homeostasis in the normal adult cartilage phenotype requires normal turnover of matrix components, principally collagen and proteoglycan. Chondrocytes and synovial fibroblasts are targeted, via specific cell-surface receptors, by cytokines like interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) to produce matrix proteases and to suppress the synthesis of collagen and proteoglycan. Thus, cytokines not only favor tissue destruction, but also inhibit tissue repair. A structurally heterogeneous group of factors capable of directly antagonizing cytokine action is described, which acts either by blocking cytokine-receptor binding, inhibiting local cytokine synthesis, or complexing the cytokine into an inactive form. Furthermore, many growth factors, such as transforming growth factor-beta (TGF-beta), can counteract the net effect of cytokines by stimulating the synthesis of matrix components or natural inhibitors of cartilage degrading enzymes.

Glucocorticoid receptors in the gill tissue of fish

The presence of glucocorticoid-binding macromolecular receptors was demonstrated in the Na2MO4 (10 mM)-stabilized gill cytosol of the American eel, Anguilla rostrata and in that of the trout, Salmo gairdneri. In all experiments, tritiated triamcinolone acetonide [( 3H]TA) was used as ligand. In the eel, the steroid was bound with a KD of 2.84 +/- 0.4 nM and an Nmax of 188 +/- 34 fmol/mg protein. The binding parameters for the trout cytosol were KD = 1.43 +/- 0.13 nM; Nmax = 271 +/- 113 fmol/mg protein. Competition studies with [3H]TA-labeled eel gill cytosol and radioinert steroids gave the following binding hierarchy: TA greater than dexamethasone greater than cortisol greater than 11-deoxycortisol greater than 21-deoxycortisol. Aldosterone, estrogens, or androgens did not complete. The eel gill receptor was deactivated by prior treatment with trypsin or mersalyl. RNase was without effect, but DNase degraded the receptor except when used in the presence of trypsin inhibitor. The eel gill TA-receptor complex sedimented on a linear (10-30%) sucrose gradient with a single peak at 7.0 S or 3.5 S, in hypotonic or hypertonic (0.4 M KCl) gradients, respectively. The eel ligand-receptor complex did not bind, following heat activation, to DNA-cellulose or phospho-cellulose, though it bound to DEAE-cellulose. In this respect, it behaved similarly to the eel intestinal mucosal TA-receptor complex, described previously. The initiation of dissociation of the eel receptor-[3H]TA complex with excess TA yielded pseudo-first-order dissociation kinetics (k-1 at 0 degree C: 2.39 X 10(-5) S-1), while the association kinetics of the receptor with the ligand was of second order (k + 1: 2.51 X 10(4) M-1 S-1). Sepharose column chromatography indicated a molecular weight of 334,690 Da. Calculation of the Stokes radius gave a value of 84.5 A and the frictional ratio, calculated from the molecular weight, was 1.84. From these data it was concluded that the gills of these two euryhaline teleosts contain tetrapod-type glucocorticoid receptors. These studies are the first to demonstrate these steroid recognition molecules in fish gill. The presence of receptors in the fish gill tissue are in agreement with the physiological action of corticosteroids in allowing adaptation of the animals to habitats of different salinity.

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

Abstract 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-β1) 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‐β 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‐β), 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‐β‐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‐β. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF‐β induction of TIMP‐3. H7 and genistein also suppressed TGF‐β‐induced TIMP‐3 protein expression. These results suggest that TGF‐β signaling for TIMP‐3 gene induction involves H7‐sensitive serine/threonine kinase as well as herbimycin A‐ and genistein‐sensitive protein tyrosine kinases. J. Cell. Biochem. 70:517–527, 1998.

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++ 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‐1β, 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‐1β 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.

Intestinal triamcinolone acetonide receptors of the eel anguilla rostrata

The binding of [6,7-3H]triamcinolone acetonide (TA) to intestinal mucosa of freshwater-adapted silver eels was studied. The cytoplasmic preparations bound the ligand with an equilibrium dissociation constant (KD) of 2.28 +/- 0.37 nM and the maximal number of binding sites (Nmax) was 960 +/- 55 fmol/mg of protein (+/- SE, n = 13). Scatchard analysis indicated the presence of a single species of binding sites. Binding was abolished following treatment of the cytosol with trypsin, N-ethylmaleimide, or Mersalyl, but DNase or RNase treatment had little effect. The competition hierarchy of radioinert steroids on the formation of the [3H]TA-receptor complex was TA greater than dexamethasone greater than cortisol greater than 11-deoxycortisol. Aldosterone, DOC, corticosterone, 11-dehydrocorticosterone, progesterone, testosterone, or estradiol-17 beta did not compete. Sedimentation of the [3H]TA-receptor complex on a linear sucrose gradient (10-30% + 10% v/v glycerol) yielded single peaks in the absence or presence of 0.4 M KCl in the gradient (6 S or 3.5 S respectively). Following heat activation the receptor-ligand complex was freely translocated to homologous nuclei in vitro, though the activated complex did not bind to DNA-cellulose. It was concluded that the eel intestinal mucosal cytosol contains a high-affinity-low capacity steroid receptor system. This is the first instance that such a system was demonstrated in fish tissue.

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 inhibitors 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.

Steroid C-20 oxidoreductase activity of duck intestinal mucosa: The interrelations of the enzymatic activity with steroid binding

The binding of corticosterone and aldosterone to domestic duck (Anas platyrhynchos)-dispersed colonic mucosal cells at 37 degrees was investigated. It was found that in contrast to experiments using cell-free intestinal preparations, corticosterone was extensively metabolized and it was the metabolite, not the native steroid that became receptor bound and all the bound ligand was in the nuclear fraction. The metabolite turned out to be identical with 4-pregnene-11 beta,20 beta, 21-triol-3-one (20 beta-dihydrocorticosterone, 20 beta-DHB). Binding experiments with [3H]corticosterone yielded the following kinetic parameters: Kd = 87.6 nM, Nmax = 337,900 sites/cell. When synthetic [3H]20 beta-DHB was used as the ligand a curvilinear-binding isotherm was obtained. This could be resolved into a high affinity-low capacity (HA) and a low affinity-high capacity (LA) component with the following binding parameters: Kd,HA = 91 nM, Nmax,HA = 130,800 sites/cell; Kd,LA = 5.4 x 10(-6) M, Nmax, LA = 3.7 x 10(6) sites/cell. Binding of the metabolite to cell-free preparations, at 0 degree, gave the following results: for cytosol, linear-binding isotherm, Kd = 14.0 nM, Nmax = 26.5 fmol/mg protein; and for crude nuclei, curvilinear-binding isotherm, Kd,HA = 45.0 nM, Nmax, HA = 5.33 pmol/mg DNA; Kd,LA = 2.2 x 10(-6) M, Nmax,LA = 286.6 pmol/mg DNA. [3H]Aldosterone was also bound by the dispersed whole cells and again, this binding was only nuclear (Kd = 9.3 nM, Nmax = 10,042 sites/cell). The bound ligand was unchanged aldosterone. Competition experiments have shown that aldosterone did not compete with 20 beta-DHB for binding sites and vice versa. The intracellular 20 beta-hydroxysteroid oxidoreductase responsible for the transformation of corticosterone was found mostly in the cytoplasm. Kinetic studies with the enzyme yielded classical Michaelis-Menten kinetics (Km = 15.7 microM, Vmax = 2.6 nmol/min/mg protein). The enzyme had an apparent Mr of 35 kDa and a Rs of 25.5 A. It is believed that our results might explain the binding of aldosterone to mineralocorticoid-binding sites in the presence of overwhelming concentrations of corticosterone and that experiments with cell-free tissue preparations, performed at 0 degree, do not reflect the true cellular-binding events.

A profile of the intestinal mucosal corticosteroid receptors in the domestic duck

The corticosteroid receptor profile of the intestinal tract of the domestic duck (maintained on either a low-sodium (LS) or a high-sodium (HS) diet) was investigated. Using tritiated triamcinolone acetonide (TA), corticosterone, or aldosterone as ligands, cytoplasmic mineralocorticoid receptors (MR, type I) and glucocorticoid receptors (GR, type II) were found in the mucosal cytosol of the jejunum and colon with the following binding parameters: LS jejunum GR-Kd, 3.4 nM; Nmax, 245 fmol/mg protein; MR-Kd, 0.54 nM; Nmax, 35 fmol/ mg protein; colon GR-3.2 nM; Nmax, 531 fmol/mg protein; MR-Kd, 0.55 nM; Nmax, 113 fmol/mg protein; HS jejunum GR--Kd, 3.2 nM; Nmax, 531 fmol/mg protein; MR--Kd, 0.30 nM; Nmax, 50 fmol/mg protein; colon GR--Kd, 1.1 nM; Nmax, 572 fmol/mg protein; MR--Kd, 0.68 nM; Nmax, 221 fmol/mg protein. The diet little influenced the GR binding parameters, while the MR (aldosterone) binding parameters showed a down-regulation following LS (high circulating aldosterone) diets. The competition hierarchy of radioinert steroids on the formation of the [3H]corticosterone-receptor complex was corticosterone = cortisol = 11-deoxycorticosterone greater than aldosterone = TA = dexamethasone much greater than 11-deoxycortisol; with [3H]aldosterone, the competition was corticosterone = progesterone = 11-deoxycorticosterone greater than aldosterone = cortisol = TA = dexamethasone greater than 11-deoxycortisol greater than 11-dehydrocorticosterone. The intestinal mucosal receptor was deactivated following treatment with trypsin. On linear sucrose gradients, receptor-ligand complexes sedimented with a single peak at 8.5 S (hypotonic gradient) and 4.0-4.5 S (hypertonic gradient), respectively. Heat-activated [3H]TA- and [3H]aldosterone-receptor complexes bound avidly to DNA-cellulose and, upon ion-exchange chromatography on DEAE-Sephacel, the presence of the negatively charged unactivated and the more positively charged activated complexes could be shown. The hydrodynamic parameters, determined by gel-filtration chromatography, gave for all three ligand-receptor complexes molecular weight values from 334,000 to 351,000 and Stokes radii from 76.8 to 80.0 A. From these studies it was concluded that the duck intestinal tract possesses vertebrate-type GR and MR, though these receptors were much less specific than their mammalian counterparts. The duck intestinal corticosteroid receptor was found to be different from those of the teleost fish and anuran amphibian, establishing the possibility of a biochemical evolution in nonmammalian intestinal corticosteroid receptor conformation.

Pregnenolone binding sites in the rat olfactory bulb

High concentrations of pregnenolone and its sulfate have been found in several areas of rat and human brain and seem to be controlled by local mechanisms. In the present experiments we have demonstrated pregnenolone binding sites in the cytosolic fraction of the rat olfactory bulb. The pregnenolone binding component showed a Kd = 2.34 +/- 0.66 x 10(-7) M and Nmax = 7.25 +/- 1.20 pmol/mg protein. Pregnenolone, pregnenolone sulfate and 17OH-pregnenolone competed equally for the binding sites while other steroids were less competitive. Protease and trypsin inhibited binding by 48 and 60% respectively. Sucrose density gradient analysis showed a minor peak at 4.6 s and a major one at 3.6 s. After gel filtration chromatography the pregnenolone binding component appeared as 2 peaks corresponding to molecular weights of approximately 150 and 220 kDa. Heating at 60 degrees C increased binding by 150%. These results indicate that the olfactory bulb pregnenolone binding component is complex in nature. Rat plasma also bound pregnenolone. Plasma binding sites could be partially differentiated from those in the olfactory bulb on the basis of susceptibility to lipoprotein lipase, effect of heating and mobility during polyacrylamide gel electrophoresis.