A p38 MAP kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA.

The mechanism by which p38 mitogen-activated protein kinase (MAPK) regulates the induction of cyclooxygenase (COX)-2 by interleukin-1 (IL-1) has been investigated in HeLa cells. SB 203580, an inhibitor of p38 MAPK, in the range 0.1-1 microM inhibited IL-1-stimulated PGE2 (but not arachidonic acid) release and this was associated with inhibition of induction of COX-2 protein and mRNA. IL-1 stimulated COX-2 transcription in HeLa cells about 2-fold as judged by both reporter gene and nuclear run-on assays. The inhibitor had no significant effect on this. However, in cells previously stimulated with IL-1 it caused rapid destabilisation of COX-2 mRNA independently of on-going transcription. The results suggest a novel function for p38 MAPK in the regulation of mRNA stability.

Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels.

The role of p38 mitogen-activated protein kinase (MAPK) in responses of human fibroblasts and vascular endothelial cells to IL-1 was investigated by use of a pyridinyl imidazole compound (SB 203580), which specifically inhibits the enzyme. SB 203580 inhibited (50% inhibitory concentration approximately 0.5 microM) IL-1-induced phosphorylation of heat shock protein 27 (an indicator of p38 MAPK activity) in fibroblasts without affecting the other known IL-1-activated protein kinase pathways (p42/p44 MAPK, p54 MAPK/c-Jun N-terminal kinase and beta-casein kinase). SB 203580 significantly inhibited IL-1-stimulated IL-6, (30 to 50% at 1 microM) but not IL-8 production from human fibroblasts (gingival and dermal) and umbilical vein endothelial cells. IL-1 induction of steady state level of IL-6 mRNA was not significantly inhibited, which is consistent with p38 MAPK regulating IL-6 production at the translational level. SB 203580 strongly inhibited IL-1-stimulated PG production by fibroblasts and human umbilical vein endothelial cells. This was associated with the inhibition of the induction of PGH synthase-2 protein and mRNA. SB 203580 also inhibited the stimulation of collagenase-1 and stromelysin-1 production by IL-1 without affecting synthesis of the tissue inhibitor of metalloproteinases (TIMP)-1. SB 203580 prevented the increase in collagenase-1 and stromelysin-1 mRNA stimulated by IL-1. In a model of cartilage breakdown, short-term IL-1-stimulated proteoglycan resorption and inhibition of proteoglycan synthesis were unaffected by SB 203580, while longer term collagen breakdown was prevented. It is concluded that 1) p38 MAPK plays an important role in the regulation of some, but not all, responses to IL-1, and 2) it is involved in the regulation of mRNA levels of some IL-1-responsive genes.

The binding subunit of pertussis toxin inhibits IL-1 induction of IL-2 and prostaglandin production.

The inhibitory effect of pertussis toxin on the action of IL-1 has been investigated. The toxin inhibited IL-1-induced production of IL-2 mRNA and protein in EL4 cells. The B oligomer of the toxin, which was shown to be devoid of ADP-ribosylating activity, proved as inhibitory as the holotoxin. The inhibition was therefore attributable to the binding subunit of the toxin and not to its ability to ADP-ribosylate G proteins. The toxin did not affect the IL-1R binding to its ligand, nor did it inhibit an early post-receptor event, the induction of the transcription factor NF kappa B. This implied that the toxin was not uncoupling IL-1R signaling. The toxin, or its B oligomer, inhibited PGE2 synthesis in human gingival fibroblasts stimulated by IL-1, but not by PMA. Assay of PG synthetic activity in the cells after addition of exogenous arachidonic acid suggested impairment by the toxin of induction of PG-synthesizing enzymes. IL-1 stimulation of IL-6 or collagenase production by fibroblasts was unaffected by pertussis toxin. The binding subunit of the toxin inhibits certain IL-1 responses by virtue of previously unrecognized actions on lymphoid and fibroblastic cells. It does not appear to block early signaling and the inhibition highly unlikely to involve inactivation of a G protein.

Pig interleukin 1. Purification of two immunologically different leukocyte proteins that cause cartilage resorption, lymphocyte activation, and fever.

Two forms of interleukin 1 (IL-1) were purified to homogeneity from the culture supernatants of pig buffy coat leukocytes stimulated with concanavalin A. The two proteins had identical Mr of 21,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but one, which had previously been purified as a cartilage-resorbing protein, had pI 5 (IL-1/5) and the other, pI 8.3 (IL-1/8). After initial gel filtration and separation by chromatofocusing IL-1/5 was purified by chromatography on hydroxyapatite and the ion exchangers, Mono S and Mono Q; IL-1/8 was purified by chromatography at pH 4.0 and pH 6.4 on Mono S. Purification was monitored by a cartilage proteoglycan release assay and both proteins had a final specific activity approximately 10(5) times that of the leukocyte culture medium. Medium conditioned by cells from 200 L of blood yielded approximately 15 micrograms of IL-1/5 and 50 micrograms IL-1/8. IL-1/8 augmented mouse thymocyte proliferation, stimulated synovial fibroblasts to produce prostaglandin E and latent collagenase, and was pyrogenic upon intracerebroventricular injection into rabbits. IL-1/5 has previously been shown to possess all these activities. An antiserum to each IL-1 was raised in rabbits. Each antiserum inhibited its respective IL-1 in a cartilage bioassay and stained it upon Western blotting. Neither antiserum inhibited or stained the other IL-1. We conclude that pig leukocytes make two immunologically distinct forms of IL-1 that have identical Mr, demonstrate the same range of biological activity, but differ in isoelectric point.

Pig catabolin is a form of interleukin 1. Cartilage and bone resorb, fibroblasts make prostaglandin and collagenase, and thymocyte proliferation is augmented in response to one protein.

Homogeneous catabolin from pig leucocytes induced proteoglycan breakdown, but not collagen breakdown, in explants of articular cartilage. It augmented lectin-induced proliferation of mouse thymocytes, stimulated production of prostaglandin E2 and collagenase by fibroblasts and chondrocytes, and increased Ca2+ release from mouse calvarial explants, all at concentrations down to 50 pM. In view of these effects it was concluded that pig catabolin is a form of interleukin 1.

Purification to homogeneity of pig leucocyte catabolin, a protein that causes cartilage resorption in vitro.

Catabolin, a protein that causes proteoglycan resorption in explants of living cartilage, was purified to homogeneity from culture medium conditioned by culturing buffy-coat leucocytes from 60 litres of pig blood in the presence of concanavalin A. The purification steps were (1) gel filtration of concentrated medium, (2) chromatofocusing, (3) hydroxyapatite chromatography, (4) anion-exchange chromatography (Mono Q), (5) reversed-phase high-pressure liquid chromatography (h.p.l.c.) (Zorbax ODS). These achieved approx. 9000-fold purification from the starting material. The purified protein when reduced ran as a single band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis with Mr 21000. On isoelectric focusing its pI was 4.8-5.0, and there was evidence of micro-heterogeneity. The protein co-migrated with active material on h.p.l.c., isoelectric focusing and SDS gels (15 and 12.5% acrylamide) under both reducing and non-reducing conditions. The pure protein caused proteoglycan release from cultured bovine nasal cartilage at 20pM. Its possible identity with interleukin 1 is discussed.

Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro.

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.

Lymphocytes induce resorption of cartilage by producing catabolin.

Mononuclear cells from pig blood, when cultured in the presence of lectins (concanavalin A or phytohaemagglutinin), release a factor that induces resorption of proteoglycan in explants of live bovine nasal cartilage. The factor had mol.wt. 20000 and pI 4.6-5.0, and was indistinguishable from the cartilage-catabolic protein catabolin isolated from culture medium of explants of pig synovial tissue. Since much of the catabolin from the mononuclear cells arose from the non-adherent population (98% lymphocytes) it was concluded that lymphocytes (and possibly monocytes) make catabolin.