Stimulation of chondrocyte-mediated cartilage destruction by S100A8 in experimental murine arthritis.

OBJECTIVE: To investigate whether S100A8 is actively involved in matrix metalloproteinase (MMP)-mediated chondrocyte activation. METHODS: S100A8 and S100A9 proteins were detected in inflamed knee joints from mice with various forms of murine arthritis, using immunolocalization. Murine chondrocyte cell line H4 was stimulated with proinflammatory cytokines or recombinant S100A8. Messenger RNA (mRNA) and protein levels were measured using reverse transcriptase-polymerase chain reaction and intracellular fluorescence-activated cell sorting (FACS). Breakdown of aggrecan on the pericellular surface of the chondrocytes was measured using VDIPEN and NITEGE antibodies and FACS, and breakdown in patellar cartilage was measured by immunolocalization. RESULTS: S100A8 and S100A9 proteins were abundantly expressed in and around chondrocytes in inflamed knee joints after induction of antigen-induced arthritis or onset of spontaneous arthritis in interleukin-1 (IL-1) receptor antagonist-knockout mice. Stimulation of chondrocytes by the proinflammatory cytokines tumor necrosis factor alpha, IL-1beta, IL-17, and interferon-gamma caused strong up-regulation of S100A8 mRNA and protein levels and up-regulation to a lesser extent of S100A9 levels. Stimulation of chondrocytes with S100A8 induced significant up-regulation of MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 mRNA levels (up-regulated 4, 4, 3, 16, 8, and 4 times, respectively). VDIPEN and NITEGE neoepitopes were significantly elevated in a concentration-dependent manner in chondrocytes treated with 0.2, 1, or 5 microg/ml of S100A8. (VDIPEN levels were elevated 17%, 67%, and 108%, respectively, and NITEGE levels were elevated 8%, 33%, and 67%, respectively.) S100A8 significantly increased the effect of IL-1beta on MMP-3, MMP-13, and ADAMTS-5. Mouse patellae incubated with both IL-1beta and S100A8 had elevated levels of NITEGE within the cartilage matrix when compared with patellae incubated with IL-1beta or S100A8 alone. CONCLUSION: These findings indicate that S100A8 and S100A9 are found in and around chondrocytes in experimental arthritis. S100A8 up-regulates and activates MMPs and aggrecanase-mediated pericellular matrix degradation.

Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis.

OBJECTIVE: To study the active involvement of Myeloid-related proteins S100A8 and S100A9 in joint inflammation and cartilage destruction during antigen-induced arthritis (AIA). METHODS: Joint inflammation and cartilage destruction was measured with 99mTc uptake and histology. The role of S100A8/A9 was investigated by inducing AIA in S100A9-/- mice that also lack S100A8 at protein level, or after intra-articular injection of rS100A8 in mouse knee joints. Cartilage destruction was measured using immunolocalisation of the neoepitope VDIPEN or NITEGE. mRNA levels of matrix metalloproteinases (MMPs) and cytokines were measured using reverse transcriptase (RT)-PCR. RESULTS: Immunisation of S100A9-/- mice with the antigen mBSA induced normal cellular and humoral responses, not different from wild type (WT) controls. However, joint swelling measured at day 3 and 7 after AIA induction was significantly lower (36 and 70%, respectively). Histologically, at day 7 AIA, cellular mass was much lower (63-80%) and proteoglycan depletion from cartilage layers was significantly reduced (between 50-95%). Cartilage destruction mediated by MMPs was absent in S100A9-/- mice but clearly present in controls. MMP3, 9 and 13 mRNA levels were significantly lowered in arthritic synovia of S100A9-/-. In vitro stimulation of macrophages by the heterodimer S100A8/A9 or S100A8 elevated mRNA levels of MMP3, 9 and in particular MMP13. Intra-articular injection of S100A8 caused prominent joint inflammation and depletion of proteoglycans at day 1. Significant upregulation of mRNA levels of S100A8/A9, cytokines (interleukin 1 (IL1)), MMPs (MMP3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4) was found in the synovium and correlated with strong upregulation of NITEGE neoepitopes within the cartilage layers. CONCLUSIONS: S100A8/A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis.

The response of the novel pro-inflammatory molecules S100A8/A9 to exercise.

Exercise shares many similarities with the acute phase response of inflammatory diseases. Recently, elevated serum levels of the novel pro-inflammatory molecules of the S100 protein family, S100A8 and S100A9, have been associated with various inflammatory diseases. The present study was conducted to assess their potential roles as inflammatory markers in monitoring the exercise-induced immune response. Seventeen male subjects of different training status performed a marathon run. Furthermore 13 subjects (10 male, 3 female) performed three different treadmill tests: strenuous (STE), moderate (MTE), and downhill (DTE). S100A8/A9 complexes were measured by ELISA, while white blood cell count (WBC) and C-reactive protein (CRP) were used as markers of the inflammatory response. Serum creatine kinase (CK) concentration was determined as a marker for muscle damage. After marathon S100A8/A9 increased dramatically during the early post-exercise period and returned to resting levels one day after the run. A similar pattern was found for WBC, while CK and CRP reached their maximum on the day after the run. Moreover, S100A8/A9 release was higher in the subgroup of well-trained athletes. The kinetic of the S100A8/A9 release after the treadmill tests depended on exercise intensity and was prolonged after eccentric exercise. In summary, the present results indicate that the novel pro-inflammatory molecules S100A8/A9 are very early and sensitive markers of the exercise-induced inflammatory response. Further investigations are necessary to evaluate the applicability of S100A8/A9 for monitoring the training process and to elucidate the dependence on training status.

Expression of the pro-inflammatory protein S100A12 (EN-RAGE) in rheumatoid and psoriatic arthritis.

OBJECTIVES: Infiltration of synovial tissue by neutrophils is crucial in rheumatoid arthritis (RA), psoriatic arthritis (PsA) and seronegative arthritis (SA). Altered vascular function and endothelial activation are important in PsA. S100A12 (EN-RAGE) is secreted by activated granulocytes and binds to the receptor for advanced glycation end products, which induces nuclear factor (NF)-kappaB-dependent activation of endothelium. METHODS: Immunohistochemical studies were performed to detect synovial S100A12 expression. We analysed serum and synovial fluid of 42 patients for S100A12 levels. RESULTS: S100A12 was strongly expressed in inflamed synovial tissue whereas it was nearly undetectable in synovia of controls or patients after successful treatment. Serum levels of S100A12 correlated with disease activity. CONCLUSIONS: Local expression of S100A12 in inflamed tissue suggests a role in synovitis, especially in PsA. High serum concentrations of S100A12 in patients with active arthritis compared with healthy controls or patients in remission point to its usefulness as a serum marker.

Myeloid-related protein (MRP)-8 from cervico-vaginal secretions activates HIV replication.

OBJECTIVES: To identify a substance found in female genital tract secretions that enhances HIV expression in infected cells. DESIGN: Cervico-vaginal lavages (CVL), collected in sterile normal saline, were fractionated and tested for HIV-inducing activity using HIV-infected monocytes. METHODS: To purify the component(s) of CVL that enhance HIV production, Mono-Q ion exchange chromatography followed by Superose-12 molecular sieve analysis, and SDS--PAGE were performed. The purified protein was identified by amino acid sequence analysis. RESULTS: SDS--PAGE of bioactive fractions showed a 14 kDa polypeptide band. Amino acid sequence analysis of selected peptides from the 14 kDa band showed 100% homology with the myeloid-related protein (MRP)-8, an inflammatory protein found in mucosal secretions. Western blot analysis revealed that bioactive CVL contained more immunoreactive MRP-8 than samples without bioactivity. The HIV-inducing activity of MRP-8 was further confirmed by showing that human recombinant MRP-8 increased HIV expression by up to 40-fold. CONCLUSIONS: MRP-8 in cervico-vaginal secretions stimulates HIV production. Strategies aimed at blocking MRP-8 activity in the genital tract could reduce risk of sexual as well as maternal--infant transmission of HIV.

Molecular analysis of the mouse S100A9 gene and evidence that the myeloid specific transcription factor C/EBPepsilon is not required for the regulation of the S100A9/A8 gene expression in neutrophils.

The genomic locus of the mouse S100A9 (MRP14) gene, a myeloid expressed gene belonging to the S100 family, is split in three exons and two introns. Insertions of B1 like and LINE elements as well as several sequence repeat structures are scattered over the gene suggesting that this region of the S100 gene cluster has been the subject of a high mutational activity in mouse evolution. The insertions may represent molecular footprints of a recently postulated inversion event, which resulted in a rearrangement of the S100 gene cluster in mouse compared to man. Deletion analysis of the promoter reveals, that a 1200 bp fragment is able to direct a cell type-specific expression of a reporter gene in granulocytic 32D cells. Unexpectedly, the myeloid-specific transcription factor C/EBPepsilon is not needed for the transcriptional upregulation of the S100A9 and S100A8 genes in neutrophils. The data described here provide further insights into the evolution of the S100 gene cluster and into the myeloid-specific regulation of the murine S100A9 gene expression.

Interaction of S100A8/S100A9-arachidonic acid complexes with the scavenger receptor CD36 may facilitate fatty acid uptake by endothelial cells.

Recently, we showed that S100A8/A9 were secreted from phorbol ester-stimulated neutrophil-like HL-60 cells, thereby carrying arachidonic acid [Kerkhoff et al. (1999) J. Biol. Chem. 274, 32672-32679]. The present study was undertaken to evaluate whether the secreted S100A8/A9-AA complex might be involved in transcellular eicosanoid metabolism. In the presence of S100A8/A9, arachidonic acid was rapidly taken up by human umbilical vein endothelial cells in a saturable and energy-dependent fashion. Protein-facilitated arachidonate uptake was confirmed by its sensitivity toward the protein modifiers bromobimane and phloretin. Both potassium and sodium depletion did not affect the arachidonate transport, indicating that arachidonate influx was independent of endocytosis. The uptake of exogenous arachidonic acid by HUVEC was predominantly mediated by FAT/CD36. This conclusion was drawn by the findings that (i) arachidonate uptake was drastically inhibited by sulfo-N-succinimidyl oleate, a specific inhibitor of FAT/CD36; (ii) the maximal inhibition of arachidonate uptake induced by SSO was similar to that effected by ATP depletion; and (iii) the arachidonate transport was 2-fold higher in COS-7 cells transfected with the pEF.BOS-CD36 expression vector than in the empty vector-transfected COS-7 cells. Kinetic studies of arachidonate uptake were indicative for an interaction between fatty acid transporter and S100A8/A9-AA complex that was confirmed by an in vitro protein-protein interaction assay. FAT/CD36 has been suggested to be involved in inflammatory responses, and S100A8/A9 are released from activated leukocytes at inflammatory loci. Therefore, it can be envisioned that their interaction might propagate host response by perpetuating recruitment and activation of cellular effectors.

Biochemical characterization of the murine S100A9 (MRP14) protein suggests that it is functionally equivalent to its human counterpart despite its low degree of sequence homology.

Due to the low degree of sequence similarity it has been speculated that murine and human S100A9 (MRP14), an inflammatory marker protein belonging to the S100 protein family, may have different cellular functions in mouse and man. The present study was undertaken to investigate the murine S100A9 protein (mS100A9) biochemically. We demonstrate that in murine peripheral CD11b+ cells up to 20% of the protein of the cytosolic fraction consists of mS100A9 and that several minor mS100A9 isoforms are present. Cell fractionation experiments with CD11b+ murine leukocytes showed that mS100A9 is found in the cytosol as well as in the insoluble fraction. Transient expression of a green fluorescence protein-mS100A9 fusion in mammalian cells revealed that mS100A9 is localized in neither the nucleus nor the vesicles. Recombinantly expressed murine S100A9 interacts in vitro with murine and human S100A8 in an in vitro glutathione S-transferase pull-down assay. Homodimerization was not observed. For further biochemical analysis the myeloid 32D cell line is presented as a suitable model, to study murine myeloid expressed S100 proteins. Both murine S100A9 and its dimerization partner mS100A8 are expressed at the onset of granulocyte-colony stimulating factor induced myeloid differentiation. Substantial amounts of this complex are constitutively secreted by granulocytic 32D cells into the medium. In summary, these data suggest, that the human and murine S100A9 may share a higher degree of functional homology than of sequence similarity.

Zinc binding reverses the calcium-induced arachidonic acid-binding capacity of the S100A8/A9 protein complex.

Analysis of the calcium-induced arachidonic acid (AA) binding to S100A8/A9 revealed that maximal AA binding was achieved at molar ratios of 1 mol S100A8 and 1 mol S100A9 and for values greater than 3 calciums per EF-hand. The AA binding capacity was not induced by the binding of other bivalent cations, such as Zn2+, Cu2+, and Mg2+, to the protein complex. In contrast, the binding of AA was prevented by the addition of either Zn2+ or Cu2+ in the presence of calcium, whereas Mg2+ failed to abrogate the AA binding capacity. The inhibitory effect was not due to blocking the formation of S100A8/A9 as demonstrated by a protein-protein interaction assay. Fluorescence measurements gave evidence that both Zn2+ and Cu2+ induce different conformational changes thereby affecting the calcium-induced formation of the AA binding pocket within the protein complex. Due to the fact that the inhibitory effect of Zn2+ was present at physiological serum concentrations, it is assumed that released S100A8/A9 may carry AA at inflammatory lesions, but not within the blood compartment.

Analysis of the MRP8-MRP14 protein-protein interaction by the two-hybrid system suggests a prominent role of the C-terminal domain of S100 proteins in dimer formation.

Calcium-binding S100 proteins are thought to play a central role in calcium-mediated signal transduction pathways. They consist of two helix-loop-helix, calcium-binding EF-hand domains. A characteristic feature is their tendency to form homo- and/or heterodimeric complexes. This report presents for the first time a functional "in vivo" approach to the analysis of S100 protein dimerization. Using the two-hybrid system we analyzed the dimerization of MRP8 (S100A8) and MRP14 (S100A9), two S100 proteins expressed in myeloid cells. It is reported that the MRP8-MRP14 heteromer is the clearly preferred complex in both man and mouse. The ability to homodimerize, however, appears to be restricted to the murine MRPs. Interaction analysis of chimeric murine/human MRP14 proteins indicates, that the C-terminal EF-hand domain plays a prominent role in MRP8-MRP14 interaction and determines the specificity of dimerization. Site-directed mutagenesis of four evolutionary conserved hydrophobic amino acids, which have been recently supposed to be essential for S100 protein dimerization, suggests that at least one of these, namely the most N-terminal located residue, is not critical for dimerization.

The gene encoding the myeloid-related protein 14 (MRP14), a calcium-binding protein expressed in granulocytes and monocytes, contains a potent enhancer element in the first intron.

Myeloid-related proteins 8 and 14 (MRP8 and MRP14) are two Ca2+-binding proteins of the S-100 family highly abundant in myelomonocytic cells. The expression is not only dependent on the developmental status of the cell but also on the inflammatory situation in the tissue. In order to identify regulatory elements responsible for the high expression of MRP14 in myeloid cells, reporter gene constructs have been transfected into HL-60 cells, Mono Mac 6 cells, and L132 cells. We demonstrated that a DNA element in the first intron (positions 153-361) enhances the transcriptional activity of the homologous promoter and of the heterologous herpes simplex virus thymidine kinase promoter up to 37-fold. To further identify the functional site, the region between positions 153 and 192 was analyzed functionally using the thymidine kinase promoter. The region increased the expression in the same magnitude as the complete intron. This enhancer is highly conserved in the human and murine MRP genes, indicative of its involvement in the transcription of MRPs. Protein binding to the region is demonstrated using EMSA, DNA cross-linking, Southwestern blotting, and affinity purification. Affinity purification confirms that four proteins bind to the enhancer element.

Members of the AAA-gene family are involved in early embryogenesis of vertebrates.

Here the nucleotide sequence of three cDNAs from Xenopus laevis are reported. The corresponding genes belong to the AAA-family of genes showing the strongest sequence homology to the human HIV-1 Tat binding protein 1 (TBP-1) and to the yeast SUG1 gene, respectively. Analysis of their expression pattern indicate that they are maternally stored in oocytes and involved in very early stages of vertebrate embryogenesis.

The heterodimer of the Ca2+-binding proteins MRP8 and MRP14 binds to arachidonic acid.

The S100 proteins MRP8 and MRP14 have been shown to be expressed by myeloid cells during inflammatory reactions. Since the majority of S100 proteins exhibit their biological activity when associated as complex it was investigated whether murine MRP8 and MRP14 form heterodimers and whether this complex may bind lipids of the cell membrane. This is of particular importance since their anchoring into the plasma membrane is unclear although upon calcium binding the proteins translocate from the cytoplasma to the cytoskeleton and the plasma membrane. Using recombinant proteins we could show that not the monomers but only the heterodimers specifically bind arachidonic acid. This finding opens new perspectives for the role of MRP8 and MRP14 in acute and chronic inflammatory processes.

Molecular characterisation of the genomic locus of the mouse MRP8 gene.

MRP8 is an inflammatory marker protein specifically expressed throughout the myeloid cell lineage in mouse and humans. Here the nucleotide sequence and the genomic structure of the mouse MRP8 gene (MM-MRP8) is presented. A strong homology between the mouse and human MRP8 promoters reflects the highly specific expression pattern of both genes and suggests that a conserved transcriptional machinery regulates these genes.

A homologue of the human MSS1 gene, a positive modulator of HIV-1 gene expression, is massively expressed in Xenopus oocytes.

Here the nucleotide sequence of a Xenopus homologue of the human MSS1 gene, a positive modulator of the HIV-1 Tat mediated transactivation in mammalian cells, is presented. This gene is highly conserved and almost exclusively expressed in Xenopus oocytes. We speculate about a possible role of this gene in the HIV-1 Tat/TAR mediated transactivation in Xenopus oocytes.

The mouse homologue of the HTLV-I tax responsive element binding protein TAXREB107 is a highly conserved gene which may regulate some basal cellular functions.

We report the cDNA sequence of a mouse gene homologous to the HTLV-I tax responsive element binding protein TAXREB107 (M-TAXREB107). This gene is constitutively and ubiquitously expressed indicating a conserved biological function. We present evidence that its transcription is under strict control of a regulatory factor, which is rapidly metabolized.

Identification of a novel HIV-1 TAR RNA bulge binding protein.

The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.

Anther-specific expression of the rolB gene of Agrobacterium rhizogenes increases IAA content in anthers and alters anther development and whole flower growth.

The promoter activity of a 2.2-kb DNA fragment from the 5' flanking sequence of the tap1 gene of snapdragon has been characterised in tobacco with the ß-glucuronidase reporter gene. The tap1 promoter conferred to the reporter gene an expression that was limited to the early stages of anther development. Expression of the rolB gene of Agrobacterium rhizogenes under the control of the tap1 promoter impaired the development of tobacco flowers. Tobacco plants transgenic for the tap1-rolB chimeric gene showed altered anthers and a reduction in whole flower growth. Such growth alterations were correlated with an increase in free IAA content and a decrease in gibberellin activity present in anthers. Since the rolB gene codes for an indole ß-glucosidase (Estruch et al. 1991), we interpret the phenotypic alterations to be a consequence of the increased content and activity of auxin in anthers. The perturbation of anther development would in turn affect gibberellin production and content, which is reflected in a reduced elongation of flowers.

The MADS box gene family in tomato: temporal expression during floral development, conserved secondary structures and homology with homeotic genes from Antirrhinum and Arabidopsis.

Five genes with homology to the floral homeotic genes deficiens of Antirrhinum and agamous of Arabidopsis were isolated from tomato. Each of the five genes is unique in the genome and could be localized to a different chromosome by RFLP mapping. Four of the tomato genes (hereafter TM) are flower-specific with distinguishable temporal expression. TM4 and TM8 are 'early', while TM5 and TM6 are 'late' genes. TM4 is homologous to squamous and TM6 is similar to deficiens, which are, respectively, 'early' and 'late' bona fide homeotic genes in Antirrhinum. The proteins encoded by the five tomato genes, like several known homeotic genes from other plants, contain within their N-terminus a highly conserved DNA-binding domain, the MADS box. All known plant MADS box genes also share, however, other properties. They all contain a central, moderately conserved, and rather basic domain, and a highly divergent or even missing C-terminal domain. Furthermore, molecular modelling predicts the presence of a conserved amphipatic alpha helix, at a constant distance from the MADS box in each of these proteins. The common properties of eight MADS box proteins from three plant families indicate that all their domains were coded for by the same ancestor gene. The sequence homology between pairs of MADS genes from different species indicates that the MADS ancestor gene multiplied and diverged in an ancestor plant common to several dicotyledon families.

Molecular characterization of two stamen-specific genes, tap1 and fil1, that are expressed in the wild type, but not in the deficiens mutant of Antirrhinum majus.

Deficiens, a homeotic gene involved in the genetic control of flower development, codes for a putative transcription factor. Upon mutation of the gene, petals are transformed to sepals and stamens to carpels, indicating that deficiens is essential for the activation of genes required for petal and stamen formation. In a search for putative target genes of deficiens, several stamen- and petal-specific genes were cloned that are expressed in wild type but not in the deficiensglobifera mutant. In this report the molecular characterization of two of these genes, tap1 and fil1, is presented. They are transiently expressed during flower development. In situ hybridization data demonstrate that tap1 is expressed in the tapetum of the anthers and fil1 in the filament of the stamen and at the bases of the petals. Both genes encode small proteins with N-terminal hydrophobic domains suggesting that they are secreted. We discuss possible functions of the gene products and their relationship to the deficiens gene.