Faculty Bibliography

Tumor necrosis factor (TNF) activates both p42 and p44 mitogen-activated protein kinases (MAPK) in human FS-4 fibroblasts, cells for which TNF is mitogenic. We now show that TNF activates p42 MAPK in two cell lines whose growth is inhibited by TNF. A mutant TNF that binds only to the p55 TNF receptor (TNFR) produced a similar degree of activation as wild-type TNF in FS-4 fibroblasts, indicating that the p55 TNFR is sufficient to mediate p42/p44 MAPK activation. The upstream intracellular signals that couple the TNFR to MAPK activation are still poorly defined. We now show that neither phorbol ester-sensitive protein kinase C nor Gialpha link TNF to p42/p44 MAPK activation, because pretreatment of FS-4 cells with phorbol ester to down-regulate protein kinase C or pretreatment with pertussis toxin to block Gialpha does not inhibit p42/p44 MAPK activation by TNF. To further analyze MAPK activation in FS-4 cells, we compared p42/p44 MAPK activation by TNF and epidermal growth factor (EGF). While tyrosine phosphorylation of p42/p44 MAPK was detected almost immediately (30 s) after stimulating cells with EGF, TNF-induced tyrosine phosphorylation was detected only after a more prolonged time interval (initially detected at 5 min and peaking at 15-30 min). In addition, the anti-inflammatory drug sodium salicylate, previously demonstrated to inhibit NF- kappaB activation by TNF, blocked the activation of p42/p44 MAPK in response to TNF but not in response to EGF. These findings demonstrate that the TNF and EGF receptors utilize distinct signaling molecules to couple to MAPK activation. Elucidation of the mechanism whereby sodium salicylate blocks TNF-induced p42/p44 MAPK activation may help to clarify TNF-activated signaling pathways

OBJECTIVE: The hyaluronan-binding protein TSG-6 (tumor necrosis factor-stimulated gene 6) forms a stable complex with the serine protease inhibitor, inter-alpha-inhibitor, potentiates the inhibition of plasmin activity, and has antiinflammatory effects in vivo. This study examines the expression of TSG-6 in human articular chondrocytes and cartilage. METHODS: Human articular chondrocytes and cartilage explants were stimulated with cytokines, growth factors, and other agents. TSG-6 expression was analyzed by imaging-assisted Northern and Western blotting. RESULT: TSG-6 messenger RNA (mRNA) expression was upregulated by cytokines and growth factors, predominantly interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), platelet-derived growth factor AA (PDGF-AA), and transforming growth factor beta 1 (TGF beta 1). TSG-6 mRNA induction by TGF beta 1 was delayed as compared with IL-1beta. Treatment of the cells with the glucocorticoid dexamethasone neither induced TSG-6 mRNA nor did it affect IL-1 beta-induced transcript levels. TSG-6 mRNA induction may involve several signal transduction pathways. The strong transcriptional stimulation by phorbol myristate acetate suggests protein kinase C (PKC)-mediated signaling. In contrast, PKA- and Ca- dependent signals are only marginally involved as messengers leading to increased TSG-6 levels after IL-1beta and TNF alpha treatment. In chondrocyte and cartilage organ cultures, both free TSG-6 (35 kd) and the complex with inter-alpha-inhibitor (120 kd) were present and upregulated by IL-1 beta, TNF alpha, or TGF beta 1. CONCLUSION: Chondrocytes are a source of TSG-6 which may play a role in cartilage remodeling and joint inflammation

A construct comprising three tandemly repeated copies of the kappaB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-kappaBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-kappaB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-kappaBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein alpha (C/EBP-alpha) (but not C/EBP-beta or C/EBP-delta). Stimulation by C/EBP-alpha required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-kappaBCAT was seen after co-transfection with C/EBP-alpha in F9 mouse embryonic carcinoma cells, known to be deficient in several transcription factors. To determine whether transcriptional activation was the result of interaction with one of the major NF-kappaB proteins, we co-transfected C/EBP-alpha with NF-kappaB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP- alpha reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a kappaB probe revealed that the addition of recombinant C/EBP-alpha protein to nuclear extracts from untreated FS-4 cells resulted in the appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-kappaB proteins had no effect. Our findings show that C/EBP-alpha may cause activation of some kappaB element-containing genes lacking C/EBP binding sites

TNF-stimulated gene 6 (tsg6), encoding a 35-kDa secretory glycoprotein (TSG-6), is induced in fibroblasts, chondrocytes, synovial cells, and mononuclear cells by the proinflammatory cytokines TNF-alpha and IL-1, or by LPS. Large amounts of TSG-6 protein were found in synovial fluids of patients with rheumatoid arthritis. TSG-6 protein forms a stable complex with components of the serine protease inhibitor, inter-alpha-inhibitor (I alpha I). In this work, we show that TSG-6 potentiates the inhibitory effect of l alpha l on the protease activity of plasmin. The plasmin/plasminogen activator system is important in the protease network associated with inflammation. To test the hypothesis that through their cooperative inhibitory effect on plasmin TSG-6 and l alpha l can modulate the protease network and thus inhibit inflammation, we examined the effect of TSG-6 on experimentally induced inflammation. Human recombinant TSG-6 protein showed a potent anti-inflammatory activity in the murine air pouch model of carrageenan- or IL-1-induced acute inflammation. The inhibitory effect of locally administered TSG-6 on the IL-1-induced cellular infiltration was comparable with that of systemic dexamethasone treatment. Two mutant TSG-6 proteins with single amino acid substitutions close to the N terminus showed a complete or partial loss of anti-inflammatory activity. The anti-inflammatory effect of the TNF/IL-1-inducible TSG-6 protein, along with its ability to inhibit protease action through interaction with l alpha l, suggests that TSG-6 production during inflammation is part of a negative feedback loop operating through the protease network

TSG-6 is an arthritis-associated hyaluronan binding protein whose production in synovial cells, chondrocytes, fibroblasts and mononuclear cells is stimulated by TNF-alpha and IL-1. The purpose of this study was to gain insights into the role of TSG-6 and its functional interactions with hyaluronan in inflammation. In the murine air pouch model of carrageenan/IL-1-induced inflammation TSG-6 showed a dramatic inhibitory effect on the cellular infiltration of the inflammatory site by neutrophilic PMN, while hyaluronan enhanced cellular infiltration. There was no indication of a neutralizing or cooperative effect when TSG-6 and hyaluronan were injected together. The potent antiinflammatory effect of TSG-6 along with its induction by proinflammatory cytokines suggests that TSG-6 is part of a negative feedback loop in the control of the inflammatory response

Human TNF-stimulated gene 14 (TSG-14) encodes a secreted 42-kDa glycoprotein that shows significant homology to proteins of the pentraxin family, which includes the acute phase reactants C-reactive protein and serum amyloid P component. Levels of TSG-14 protein (also termed PTX-3) become elevated in the serum of mice and humans after injection with bacterial lipopolysaccharide, but in contrast to conventional acute phase proteins, the bulk of TSG-14 synthesis in the intact organism occurs outside the liver. In the present study we cloned and partially sequenced murine genomic TSG-14 DNA. Analysis of the coding region predicts a high degree of amino acid sequence homology between murine and human TSG-14 (88 and 75% identity in the first and second exons, respectively). The promoter of the TSG-14 gene lacks consensus sequences for either a TATA box or CCAAT box. Primer extension analysis and S1 nuclease protection assay revealed one major transcription start site, situated within a consensus sequence for an initiator element. Sequence analysis of a approximately 1.4-kilobase pair fragment of the 5'-flanking region of the TSG-14 gene revealed the presence of numerous potential enhancer binding elements, including six NF-IL6-like sites, four AP-1, one AP-2, one NF-kB, two Sp1, two interferon-gamma-activated sites (GAS), one Hox-1.3, and five binding sites for Ets family members. Transfection of BALB/c 3T3 cells with promoter DNA fragments linked to the luciferase reporter gene revealed that the 5'-flanking region of the TSG-14 gene comprises elements that can mediate a basal level of transcription and inducibility by TNF

We have compared the regulation of the human metallothionein (MT)-IIA gene by the cytokines tumour necrosis factor-alpha (TNF) and interferon beta (IFN-beta) in human fibroblasts. Both TNF and IFN-beta induced MT-II mRNA rapidly, but stimulation by TNF was more sustained. The effects of TNF and IFN-beta were further distinguished by the action of the protein synthesis inhibitor cycloheximide, which reduced MT-II mRNA stimulation by TNF but enhanced IFN-beta-induced MT-II mRNA. These results suggested that TNF and IFN-beta activate MT-II gene expression by partially distinct mechanisms. Consistent with this notion, combined treatment with both cytokines resulted in more than an additive level of MT-II mRNA induction. TNF and IFN-beta also acted cooperatively in inducing MT-II mRNA in HeLa cells. A reporter construct containing positions -765/+80 of the MT-II promoter linked to the CAT reporter gene failed to respond to either TNF or IFN-beta in HeLa cells, despite the presence of a putative IFN-stimulated response element (ISRE) and an activator protein-1 (AP-1) binding site, suggesting that these elements are insufficient for the activation of the MT-II gene by these cytokines. Thus induction of MT-II expression differs from the genes whose activation by TNF can be induced via the AP-1 element alone, as well as those genes whose activation by IFN is mediated solely through the ISRE site

Tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) encodes a protein expressed during inflammation. We have previously shown that transcription factors of the NF-IL6 and AP-1 families cooperatively modulate activation of the TSG-6 gene by TNF or interleukin 1 (IL-1) through a promoter region that contains an NF-IL6 site (-106 to -114) and an AP-1 element (-126 to -119). In this study we report the identification of an additional NF-IL6 site (NF-IL6*) located at positions -92 to -83. Footprinting and electrophoretic mobility shift assay suggested that NF-IL6 binds with higher affinity to the newly identified NF-IL6* site than to the earlier identified promoter-distal NF-IL6 site and that the two sites cooperate in binding NF-IL6. TNF and IL-1 stimulate specific binding of nuclear proteins to the NF-IL6* site more efficiently than to the promoter-distal NF-IL6 site. Moreover, a mutation in the NF-IL6* site abolished transactivation of the TSG-6 promoter by NF-IL6 despite the presence of the intact promoter-distal NF-IL6 site. A mutation in the promoter-distal NF-IL6 site also greatly decreased activation of the TSG-6 promoter by NF-IL6. We conclude that the two NF-IL6 sites are functionally interdependent in the activation of the TSG-6 gene