Interleukin 1 induces hypoxia-inducible factor 1 in human gingival and synovial fibroblasts.

Rheumatoid arthritis and periodontitis are inflammatory diseases modulated by proinflammatory cytokines [e.g. interleukin (IL-1) 1 and tumour necrosis factor alpha], which activate local fibroblasts to do the following: (1) proliferate, (2) induce gene expression and (3) produce destructive metalloproteinases. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor (composed of HIF-1alpha and HIF-1beta/aryl hydrocarbon receptor nuclear transporter) that is modulated by hypoxia. HIF-1 binds to and induces several genes containing an HIF-1 consensus-binding site, including vascular endothelial growth factor and several glycolytic enzymes. Through differential screening of a human synovial fibroblast cDNA library, we identified HIF-1alpha as a clone up-regulated by IL-1. The mRNA for HIF-1alpha subunit was increased 3-4-fold by Northern blot analysis after cells had been incubated for 3 h in the presence of IL-1. In addition, IL-1 increased the binding of the heterodimer HIF-1 to the HIF consensus sequence. These results suggest that HIF-1 might have a role in inflammation, possibly in attempting to re-establish homoeostasis.

Osteopathic family physicians' attitudes, knowledge, and self-reported practices regarding obesity.

The study's purpose was to examine attitudes, knowledge, and self-reported practices of osteopathic family physicians regarding obesity. A survey was developed and administered to osteopathic family physicians. Respondents were predominantly male (76%), and their mean age was 44.6 years. Most had been in practice for fewer than 10 years. Physicians surveyed used diet, exercise, and behavior modification most often in treating obese patients. They were reluctant to suggest medications for treatment of obesity. The physicians also displayed a lack of understanding of the proper assessment of obese patients. These family physicians admitted that medical journals and continuing education were their major sources of information about obesity. Most agreed that their medical school training was deficient in regard to the treatment of obesity, but a majority wished further training. Most of the physicians surveyed had a desire to collaborate on obesity-related research projects. These trends appeared not to be related to physician gender, age, years in practice, or weight. The results of this survey indicated that osteopathic family physicians desire to be updated on current research regarding obesity and use of interdisciplinary modalities. Additional access to training could make an impact on these attitudes and thereby influence their patient management.

Identification of a cytokine-induced repressor of interleukin-1 stimulated expression of stromelysin 1 (MMP-3).

Stromelysin 1 (MMP-3) is a matrix metalloproteinase with broad substrate specificity that has been linked to joint and tissue destruction associated with chronic inflammatory diseases such as rheumatoid arthritis and periodontitis. Transcription of the stromelysin gene is induced by inflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor as well as a number of other cytokines and mitogens, but the exact mechanisms involved in its regulation are not fully understood. To identify transcription factors and cis elements potentially involved in the IL-1 induction of stromelysin, the human stromelysin 5'-flanking region was screened by electrophoretic mobility shift assay for IL-1-induced DNA-binding complexes in human synovial and gingival fibroblasts. Here we report the identification of such a complex binding to the region -1614 to -1595 (5'-G(T)TTTTTCCCCCCATCAAAG-3') termed the stromelysin IL-1 responsive element site. Binding to this site is also induced by tumor necrosis factor but not by platelet-derived growth factor or interleukin 4. UV cross-linking demonstrates that there are at least two DNA-binding proteins involved, of approximately 48 and 52 kDa. Transient transfection experiments in human foreskin fibroblasts demonstrate that proteins binding to this site act as a repressor of IL-1-induced expression of the stromelysin gene.

Induction of mitogen-inducible nuclear orphan receptor by interleukin 1 in human synovial and gingival fibroblasts.

High levels of interleukin 1 (IL-1) found in inflammatory diseases such as rheumatoid arthritis and periodontitis act on the local fibroblasts, resulting in an altered phenotype characterized by hyperplasia and the production of inflammatory mediators and destructive enzymes. The goal of this study was to identify genes induced as an early response to IL-1 in synovial and gingival fibroblasts which might play a regulatory role in the cascade of events leading to their activation. Using the technique of mRNA differential display, we have identified the mitogen-inducible nuclear orphan receptor (MINOR) as a gene up-regulated by IL-1 in human synovial and gingival fibroblasts. The rapid induction of both mRNA and DNA binding activity suggests that MINOR may play an important early role in regulating the response of fibroblasts to inflammation.

Interleukin-4 suppression of interleukin-1-induced transcription of collagenase (MMP-1) and stromelysin 1 (MMP-3) in human synovial fibroblasts.

OBJECTIVE: To determine the effects of interleukin-4 (IL-4) on IL-1 induction of collagenase (matrix metalloproteinase 1 [MMP-1]) and stromelysin-1 (MMP-3) in human synovial fibroblasts. METHODS: Northern blot analysis was performed to determine the effects of IL-4 on IL-1 induction of MMP messenger RNA (mRNA). MMP protein levels were determined by enzyme-linked immunosorbent assay, and prostaglandin E2 (PGE2) levels were measured by enzyme immunoassay. Run-on transcription assays and transient transfection experiments were performed to determine whether the effects of IL-4 occur at the level of transcription. Activator protein 1 (AP-1) binding was assessed by electrophoretic mobility shift assay. RESULTS: Northern blot analysis revealed that coincubation of synovial fibroblasts with IL-1 and IL-4 resulted in a significant decrease in both collagenase and stromelysin mRNA levels compared with IL-1 alone, with a concomitant decrease in MMP protein levels. This inhibition is dose dependent, with an IC50 (50% inhibition concentration) for both MMPs of approximately 0.3 ng of IL-4 per ml, and is at least somewhat selective, since IL-1 induction of c-fos mRNA is not affected. Nuclear run-on experiments and transient transfection studies demonstrated that the suppression of IL-1-induced collagenase and stromelysin expression by IL-4 occurs at least in part at the transcriptional level, and that binding of transcription factor AP-1 is not affected. Although IL-1-induced levels of PGE2 are reduced by IL-4, exogenous addition of PGE2 does not abrogate the inhibitory effects of IL-4 on MMP expression. CONCLUSION: IL-4 inhibits IL-1 induction of both collagenase and stromelysin, as well as PGE2 production, in human synovial fibroblasts. The inhibition occurs at least in part at the level of transcription, does not affect binding of transcription factor AP-1, and appears to involve a mechanism that is independent of the ability of IL-4 to inhibit production of PGE2.

Induction of bone morphogenetic protein-2 by interleukin-1 in human fibroblasts.

Rheumatoid arthritis and periodontitis are chronic inflammatory diseases associated with tissue destruction that is mediated in part by elevated levels of cytokines (e.g., interleukin-1 and tumor necrosis factor). Differential screening of a human synovial fibroblast cDNA library for interleukin-1 induced genes revealed a clone identical to the gene encoding human bone morphogenetic protein-2. Northern blot analysis of human synovial fibroblast mRNA confirmed up-regulation of bone morphogenetic protein-2 in the presence of interleukin-1. Utilizing a specific antibody, levels of bone morphogenetic protein-2 protein in conditioned medium from synovial fibroblasts were also up-regulated in the presence of interleukin-1. This is the first report of the production of bone morphogenetic protein-2 by synovial fibroblasts, and the first report of its up-regulation in response to interleukin-1. However, interleukin-1 did not induce bone morphogenetic protein-2 mRNA in human gingival fibroblasts.

Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation.

Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation.

Suppression of human synovial fibroblast 85 kDa phospholipase A2 by antisense reduces interleukin-1 beta induced prostaglandin E2.

OBJECTIVE: To evaluate the relative roles of rheumatoid synovial fibroblast phospholipases A2 (PLA2) in interleukin- 1beta (IL-1 beta) stimulated prostaglandin E2 (PGE2) production. METHODS: The role of the cytosolic 85 kDa PLA2 in IL-1beta induced human rheumatoid synovial fibroblast PGE2 formation was directly evaluated using an antisense phosphorothioate oligonucleotide to the initiation site of the 85 kDa PLA2 mRNA. Contribution of the 14 kDa PLA2 was assessed using selective inhibitors or a neutralizing monoclonal antibody (Mab). RESULTS: Antisense, but not sense, decreased IL-1beta upregulation of 85 kDa PLA2 activity and protein levels. The antisense effect was specific, since it did not affect 14 kDa PLA2 activity released into the media or induced cyclooxygenase II protein levels over 24 h. Antisense, but not sense, reduced PGE2 formation in a concentration dependent manner. IL-1 beta significantly upregulated cell associated 14 kDa PLA2 and its subsequent release. Specific inhibition of this enzyme by a neutralizing Mab or selective inhibitors of 14 kDA PLA2 activity did not alter IL-1beta induced PGE2 levels. CONCLUSION: These data directly support a role for the 85 kDa PLA2, but not the 14 kDa PLA2, in IL- 1beta stimulated PGE2 production from human rheumatoid synovial fibroblast.

Molecular cloning and sequencing of a human cDNA encoding ornithine decarboxylase antizyme.

We report the cloning of a cDNA encoding the human homolog of ornithine decarboxylase antizyme from a human gingival fibroblast cDNA library. The human antizyme is 84% identical to the rat sequence and shows almost no homology to the E. coli antizyme. Northern analysis studies show that this gene is expressed in both human gingival and synovial fibroblasts.

Mechanistic features associated with induction of metalloproteinases in human gingival fibroblasts by interleukin-1.

Human gingival fibroblasts were treated with recombinant interleukin-1 (IL-1) to determine the effect of this stimulus on the relative expression of collagenase (MMP-1), stromelysin (MMP-3) and plasminogen activator (PA) mRNA. The steady-state mRNA levels for these genes were determined on Northern blots. IL-1 induced steady-state levels of these mRNAs to different extents. Nuclear run-on transcription studies showed that IL-1 induction of neutral metalloproteinase may be transcriptionally regulated. Actinomycin D and protein kinase inhibitors decreased the mRNA production for all three metalloproteinases, whereas cycloheximide decreased the production of collagenase and stromelysin mRNA. Protein kinase inhibitors (H7/H8) decreased production of the three mRNAs to different extents. This study demonstrates a potentially important role for IL-1 in the regulation of metalloproteinase expression in human gingival fibroblasts. The ability of IL-1 to induce the expression of stromelysin, collagenase and PA may define a pivotal role for this cytokine in the pathogenesis of periodontitis.

Interleukin-1 selectively potentiates bradykinin-stimulated arachidonic acid release from human synovial fibroblasts.

Exposure of human synovial fibroblasts prelabelled with [3H]arachidonic acid to bradykinin causes a rapid and sustained increase in arachidonic acid release, a transient increase in cytosolic calcium and an increase in radiolabelled diacylglycerol. Activation of arachidonic acid release by bradykinin was potentiated by interleukin-1 added either simultaneously with bradykinin or to cultures 24 h before addition of bradykinin. In contrast, interleukin-1 did not modify bradykinin-induced increases in cytosolic calcium or diacylglycerol. The stimulation of arachidonic acid release in response to bradykinin, in the absence or presence of interleukin-1, was not affected by RHC-80267, an inhibitor of diacylglycerol kinase, suggesting that deacylation of diacylglycerol was not an important pathway of arachidonic acid production in cultures exposed to bradykinin. This conclusion is supported by the observation that increased release of arachidonic acid was not accompanied by increased release of [14C]stearic acid in cultures labelled with both isotopes. Bradykinin-stimulated release of arachidonic acid was prevented by down-regulating protein kinase C by pretreatment with phorbol 12-myristate 13-acetate and was unaffected by inhibitors of protein synthesis actinomycin D or cycloheximide. On the other hand, interleukin-1 amplification of bradykinin-stimulated release of arachidonic acid was blocked by actinomycin D and cycloheximide. The results from this study point to activation of phospholipase A2 as the source of arachidonic acid in response to bradykinin. Our data further indicate that interleukin-1 selectively potentiates bradykinin activation of a phospholipase A2 by a mechanism requiring protein synthesis, but has no effect on bradykinin activation of phospholipase C.

Modulation of synovial fibroblast plasminogen activator and plasminogen activator inhibitor production by protein kinase C.

Phorbol myristate acetate (PMA) added to human synovial fibroblast cultures caused a dose-dependent increase in the production of plasminogen activator inhibitor-type 1 (PAI-1). In addition, PMA inhibited endogenous and interleukin-1 (IL-1) induced plasminogen activator (PA) activity, while increasing mRNA PAI-1 levels. Other protein kinase C (PKC) activators, mezerein and teleocidin B4, caused similar effects. The simultaneous addition of the PKC antagonists, H-7 or staurosporine, prevented the inhibition of PA activity by PMA. This study shows that activation of PKC inhibits PA and stimulates PAI production in human synovial fibroblasts. These results suggest that activation of PKC may play an important role in regulating increased PA production associated with joint destruction in rheumatoid arthritis (RA).

Interleukin-1 stimulation of arachidonic acid release from human synovial fibroblasts; blockade by inhibitors of protein kinases and protein synthesis.

Addition of IL-1 (interleukin-1) to human synovial fibroblasts radiolabelled with [3H]arachidonic acid caused a linear dose-dependent increase in arachidonic acid release and a transient rise in labelled diacylglycerol. Protein kinase C activators PMA 4-phorbol 12-myristate 13-acetate and DiC8 (1,2-dioctanoyl-sn-glycerol) also increased arachidonic acid release, but the time course observed with PMA was different from that of IL-1. When cultures were treated with PMA for 16-24 h to down regulate protein kinase C, the ability of IL-1 to increase arachidonic acid release persisted to the same extent as in nontreated cultures. In contrast, PMA pretreatment prevented the eight-fold stimulation of arachidonic acid release in response to PMA observed in cultures not previously exposed to PMA. To examine the role of other kinases in IL-1 stimulated arachidonic acid release, cultures were treated with H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine dichloride), H-8 (N-[2-(methylamino) ethyl]-5-isoquinolinesulphonamide dichloride), HA1004 (N-(2-guanidoinoethyl)-5-isoquinolinesulphonamide hydrochloride), and staurosporine. IL-1 stimulation of arachidonic acid release was blocked by H-7, H-8 and staurosporine. H-7 was a more potent inhibitor than H-8, suggesting that cAMP dependent kinase did not mediate IL-1 action. Addition of H-7 at various times following IL-1 decreased IL-1 stimulated arachidonic acid release, suggesting that continued protein kinase activity was necessary for IL-1 action. Cycloheximide and actinomycin D inhibited the stimulation of arachidonic acid release by IL-1, PMA or DiC8. The addition of cycloheximide or actinomycin D 15-45 min after IL-1 also inhibited IL-1 stimulated arachidonic acid release, indicating that continued protein synthesis was required for IL-1 action. These results suggest that IL-1 stimulation of acylhydrolyase activity in human synovial cells occurs by a mechanism requiring continued protein synthesis and protein kinase activity and that neither protein kinase C nor cAMP dependent protein kinase is involved.

Interleukin-1 mediated signal transduction associated with synovial cell activation.

The early biochemical events that link interleukin-1 (IL-1) receptor occupancy to neutral proteinase production in synovial cells were studied. Addition of human r-IL-1 to human synovial cells in culture stimulated phospholipase A2 (PLA2) activity, inositol triphosphate production and plasminogen activator (PA) activity in a dose dependent manner with similar EC50 values (0.1-0.5 nM). These results, coupled with time courses and other studies, suggest that the IL-1 modulation of PA involves both products of PLA2 and phospholipase C (PLC) activation. On the other hand, the IL-1 induction of collagenase may primarily involve PLC and protein kinase C activation.

Interleukin-1 activates phospholipase A2 in human synovial cells.

Interleukin-1 (IL-1) treatment of synovial cells from rheumatoid arthritis and osteoarthritis patients resulted in a dose-dependent secretion of phospholipase A2 (PLA2). IL-1 also stimulated prostaglandin E2 and plasminogen activator synthesis, in parallel with PLA2 activation; all 3 were detectable within 6 hours of IL-1 treatment and peaked by 24 hours. Synovial cell PLA2 required calcium (5 mM) and a neutral pH (7.5) for maximal activity and appears similar to the PLA2 in synovial fluid, which has been described previously. We conclude that PLA2 can be induced by IL-1, and its secretion may contribute significantly to the inflammatory actions of IL-1.