Increased serum ß2-microglobulin is associated with clinical and immunological markers of disease activity in systemic lupus erythematosus patients.

The objective of this study was to explore the relationship between serum levels of ß2-microglobulin (ß2MG), which some studies suggest reflect disease activity in systemic lupus erythematosus (SLE), and various clinical and immunological markers of disease activity in SLE. Twenty-six SLE patients and 10 healthy controls were included. Disease activity was assessed by: SLEDAI, 24 hr-proteinuria, circulating levels of complement C3, anti-double-stranded DNA (anti-dsDNA), ß2MG and various pro-inflammatory and anti-inflammatory cytokines (IL-6, IL-8, IL-10, IL-18) measured with a multiplex assay, IFN-α assessed with a reporter gene assay, and a combined expression score of 12 IFN-α inducible genes in peripheral blood mononuclear cells. Median serum levels of ß2MG were significantly higher in SLE patients vs controls (2.8 mg/L, range: 1.1-21.6 and 1.2 mg/L, range: 0.9-1.7, respectively, p < 0.001). ß2MG was correlated with SLEDAI score (R = 0.68, p < 0.001), 24 hr-proteinuria (R = 0.64, p < 0.001), and complement C3 (R = -0.52, p = 0.007). The cytokines were significantly correlated with ß2MG: IL-6 (R = 0.45, p = 0.02), IL-8 (R = 0.75, p < 0.001), IL-10 (R = 0.67, p < 0.001) and IL-18 (R = 0.71, p < 0.001) as were serum IFN-α (R = 0.45, p = 0.02) and the IFN-α inducible gene-score (R = 0.51, p = 0.01). The results support that ß2MG may serve as a marker of disease activity in SLE. The correlations with the measured cytokines indicate that increased ß2MG in SLE reflects immunological activity.

Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDA-MB-231 cells.

BACKGROUND: Factor VIIa (FVIIa) binding to tissue factor (TF) induces cell signaling via the protease activity of FVIIa and protease-activated receptor 2 (PAR2). OBJECTIVE: We examined how the gene-expression profile induced by FVIIa corresponds to the profiles induced by protease-activated receptor 1 (PAR1) or PAR2 agonists using MDA-MB-231 breast carcinoma cells that constitutively express TF, PAR1 and PAR2. RESULTS AND CONCLUSIONS: Out of 8500 genes, FVIIa stimulation induced differential regulation of 39 genes most of which were not previously recognized as FVIIa regulated. All genes regulated by FVIIa were similarly regulated by a PAR2 agonist peptide confirming FVIIa signaling via PAR2. An appreciable fraction of the PAR2-regulated genes was also regulated by a PAR1 agonist peptide suggesting extensive redundancy between FVIIa/PAR2 signaling and thrombin/PAR1 signaling. The FVIIa regulated genes encode cytokines, chemokines and growth factors, and the gene repertoire induced by FVIIa in MDA-MB-231 cells is consistent with a role for TF-FVIIa signaling in regulation of a wound healing type of response. Interestingly, a number of genes regulated exclusively by FVIIa/PAR2-mediated cell signaling in MDA-MB-231 cells were regulated by thrombin and a PAR1 agonist, but not by FVIIa, in the TF-expressing glioblastoma U373 cell line.

Association of aldose reductase gene Z+2 polymorphism with reduced susceptibility to diabetic nephropathy in Caucasian Type 1 diabetic patients.

AIMS: The Z-2 allele of the (AC)n polymorphism in the aldose reductase gene (ALR2) confers increased risk of microvascular diabetic complications, whereas the Z+2 allele has been proposed to be a marker of protection. However data are conflicting. Therefore, we investigated whether this polymorphism is associated with diabetic nephropathy and retinopathy in Type 1 diabetes mellitus in a large case-control study and a family-based analysis. METHODS: A total of 431 Type 1 diabetic patients with diabetic nephropathy and 468 patients with longstanding Type 1 diabetes and persistent normoalbuminuria were genotyped for the case-control study. In addition, 102 case trios and 98 control trios were genotyped for a family-based study. RESULTS: Thirteen different alleles were identified. In the case-control study, the Z+2 allele frequency was significantly higher in the normoalbuminuric diabetic than in patients with diabetic nephropathy (0.17 vs. 0.11, P = 0.008), suggesting a protective function of the Z+2 allele. No significant increase in the frequency of the putative risk allele Z-2 was found in patients with diabetic nephropathy vs. controls (0.39 vs. 0.36). No association with diabetic retinopathy was found. Although the results of the transmission of the Z-2 and Z+2 alleles in the independent family-based study were consistent with the association study, the number of informative families was limited and thus differences were not statistically significant. CONCLUSIONS: The Z+2 allele of the ALR2 promoter polymorphism is associated with a reduced susceptibility to diabetic nephropathy in Danish Type 1 diabetic patients, suggesting a minor role for the polyol pathway in the pathogenesis of diabetic kidney disease. No association of the ALR2 polymorphism with diabetic retinopathy was found.

Identification of hepatic transcriptional changes in insulin-resistant rats treated with peroxisome proliferator activated receptor-alpha agonists.

Peroxisome proliferator activated receptor (PPAR)-alpha controls the expression of multiple genes involved in lipid metabolism, and activators of PPAR-alpha, such as fibrates, are commonly used drugs in the treatment of hypertriglyceridemia and other dyslipidemic states. Recent data have also suggested a role for PPAR-alpha in insulin resistance and glucose homeostasis. In the present study, we have assessed the transcriptional and physiological responses to PPAR-alpha activation in a diet-induced rat model of insulin resistance. The two PPAR-alpha activators, fenofibrate and Wy-14643, were dosed at different concentrations in high-fat fed Sprague-Dawley rats, and the transcriptional responses were examined in liver using cDNA microarrays. In these analyses, 98 genes were identified as being regulated by both compounds. From this pool of genes, 27 correlated to the observed effect on plasma insulin, including PPAR-alpha itself and the leukocyte antigen-related protein tyrosine phosphatase (PTP-LAR). PTP-LAR was downregulated by both compounds, and showed upregulation as a result of the high-fat feeding. This regulation was also observed at the protein level. Furthermore, downregulation of PTP-LAR by fenofibric acid was demonstrated in rat FaO hepatoma cells in vitro, indicating that the observed regulation of PTP-LAR by fenofibrate and Wy-14643 in vivo is mediated as a direct effect of the PPAR agonists on the hepatocytes. PTP-LAR is one of the first genes involved in insulin receptor signaling to be shown to be regulated by PPAR-alpha agonists. These data suggest that factors apart from skeletal muscle lipid supply may influence PPAR-alpha-mediated amelioration of insulin resistance.

Identification of a novel integral plasma membrane protein induced during adipocyte differentiation.

Adipocyte differentiation is co-ordinately regulated by several transcription factors and is accompanied by changes in the expression of a variety of genes. Using mRNA differential display analysis, we have isolated a novel mRNA, DD16, specifically induced during the course of adipocyte differentiation. DD16 mRNAs are present in several tissues, but among the tissues tested, a remarkably higher level of expression was found in white adipose tissue. The DD16 cDNA encoded a polypeptide of 415 amino acids containing a single N-glycosylation site and an N-terminal hydrophobic stretch of 19 amino acids forming a transmembrane segment, indicating that DD16 is a glycosylated membrane-bound protein. Polyclonal antibodies raised against the DD16 peptide detected immunoreactive DD16 in membrane fractions, notably the plasma membrane. Association of DD16 with the plasma membrane was further confirmed by biotinylation studies of cell surface proteins, suggesting that DD16 is an integral plasma membrane protein. Therefore we propose to give DD16 the name APMAP (Adipocyte Plasma Membrane-Associated Protein). Although the biological function of this polypeptide is presently unknown, our data suggest that APMAP may function as a novel protein involved in the cross-talk of mature adipocytes with the environment.

The transcription factor Fos-related antigen 1 is induced by thiazolidinediones during differentiation of 3T3-L1 cells.

Thiazolidinediones (TZDs) are a new class of compounds that improve the insulin sensitivity in patients with non-insulin-dependent diabetes mellitus (NIDDM) as well as in rodent models of NIDDM. These compounds act as high-affinity ligands for a member of the nuclear hormone receptor superfamily PPARgamma, which has been shown to play an important role in adipocyte differentiation. The strong correlation between the antidiabetic activity of TZDs and their ability to activate PPARgamma has led to suggestions that PPARgamma or downstream regulated genes mediate the effects of TZDs. To identify novel genes that potentially mediate the effects of TZDs, we have isolated genes that are differentially expressed during thiazolidinedione-stimulated differentiation of 3T3-L1 cells. Using mRNA differential display, we have compared 3T3-L1 cells treated to differentiate in the presence of BRL49653 with untreated 3T3-L1 cells and identified Fos-related antigen 1 (Fra-1), a member of the Fos protein family, as a novel molecular target for BRL49653 action in 3T3-L1 cells. Analysis of all members of the Fos-Jun family of transcription factors showed that Fra-1 was the only member that was specifically up-regulated by BRL49653. The only other member of the Fos-Jun family expressed in differentiated 3T3-L1 cells was JunD and a complex of Fra-1 and JunD was formed on a consensus AP-1 binding element in differentiated 3T3-L1 cells, suggesting that the complex of Fra-1 and JunD may play a role in the stimulation of the differentiation process of 3T3-L1 cells observed after treatment of the cells with insulin sensitizers.

Modulation of metabolism through transcriptional control has created new treatment opportunities for type 2 diabetes.

The discovery of the important metabolic and physiological role played by a family of transcription factors, the peroxisome proliferator activated receptors (PPAR), has opened up for a new understanding of the mode of action for the lipid lowering drugs known as fibrates and for the new glucose lowering compounds described as insulin sensitizers. Both of these classes of compounds have demonstrated significant efficacy in both animal models of the metabolic derangements characteristic for type 2 diabetes and in human clinical studies. The recognition of the role of these drugs as ligands for PPAR transcription factors and the development of new molecular and cellular tools to select and characterise new PPAR selective compounds will open up for the development of even better new drug candidates for the treatment of metabolic disorders associated with type 2 diabetes. With the combined strength of new transcriptional mapping technologies developed in the field of molecular biology, such as differential mRNA display and DNA microarray hybridisations, it will be possible to perform a detailed molecular characterisation of the transcriptional events involved in drug actions in cellular and tissue systems, and information gathered from such types of analysis will lead to an enormous amount of data, from which detailed knowledge of drug actions at the gene regulatory level will emerge.

U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction.

Splicing of mRNA precursors (pre-mRNAs) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use for the interaction between U2 snRNP and the branchpoint is unclear, and, in particular, none of the known mammalian factors required for this step have the sequence characteristics of proteins that hydrolyze ATP. Entry of U2 snRNP into the spliceosome is initiated by interaction of the essential splicing factor U2AF65 with the pre-mRNA polypyrimidine tract. In this report we identify a new region of U2AF65 required for function, and use this information to clone a human 56-kD U2AF65 associated protein (UAP56). We show that UAP56 is an essential splicing factor, which is recruited to the pre-mRNA dependent on U2AF65, and is required for the U2 snRNP-branchpoint interaction. The sequence of UAP56 indicates it is a member of the DEAD box family of RNA-dependent ATPases, which mediate ATP hydrolysis during several steps of yeast pre-mRNA splicing. Our results reveal a new function of U2AF65: to position a DEAD box protein required for U2 snRNP binding at the pre-mRNA branchpoint region.

Assignment of the human tryptophanyl-tRNA synthetase gene (WARS) to chromosome 14q32.2 --> q32.32.

Tryptophanyl-tRNA synthetase catalyzes the aminoacylation of tRNAtrp with tryptophan, an essential function in the cell's protein synthesis machinery. It has been shown that tryptophanyl-tRNA synthetase is induced by interferon, and this has led to hypotheses about other possible functions of tryptophanyl-tRNA synthetase. We have mapped a cDNA probe of the tryptophanyl-tRNA synthetase gene (WARS) by a combination of somatic cell hybrid analysis, fluorescence in situ hybridization (FISH), and linkage analysis. Both FISH and linkage analysis independently supported a more distal position of WARS than had been previously reported. FISH mapping indicated a most likely location at 14q32.31. Linkage analysis was based on the 40 reference families from the CEPH collaboration and resulted in a 13-point map, placing WARS, with odds of more than 1,000:1, within an area of approximately 10 cM and, with odds of 198:1, in an approximately 6 cM interval between pCMM101 and D14S27. The study provides additional integration of the physical and genetic maps of the distal part of 14q, as well as genetic tools enabling a more complete understanding of the function of tryptophanyl-tRNA synthetase, especially with regard to the cryptic inducibility of interferon.

Differential regulation of the human, interferon inducible tryptophanyl-tRNA synthetase by various cytokines in cell lines.

Cytokines regulate the expression of specific sets of proteins which mediate their biological effects. We have comprehensively delineated the regulation of the human tryptophanyl-tRNA synthetase (hWRS) by eight different cytokines (including IFNs) and poly(I).poly(C) in several cell lines. Six non-lymphoid cell lines were tested, and all of these produced human, IFN inducible hWRS (gamma 2) mRNA upon stimulation with IFN-gamma. In all these cell lines the level of gamma 2 mRNA increased 2-4 h after induction reaching a stable plateau after 8-12 h. The IFN-gamma induction of gamma 2 mRNA could be blocked by cycloheximide in human amniotic (AMA) cells, epithelial HeLa cells and HT1080 fibroblasts, but not in T98G glioblastoma cells. IFN-alpha and poly(I).poly(C) elicited small, transient gamma 2 responses in a few of the non-lymphoid cell lines, whereas none of the other six cytokines tested elicited a response. The six lymphoid cell lines tested did not show the same induction pattern. In the monocytic cells, THP-1, gamma 2 mRNA was highly induced by IFN-gamma, whereas in the B-cell line, Daudi, gamma 2 mRNA was transiently induced by IFN-alpha and poly(I).poly(C), and not by IFN-gamma. Altered mRNA turnover rate as a consequence of IFN-gamma treatment did not appear to play a significant role in the accumulation of gamma 2 transcript, since the stability essentially was the same in induced versus non-induced cells. We conclude that the hWRS gene is induced preferentially by IFN-gamma, and that the induction pattern resembles the one reported for the IFN induced enzyme, indoleamine 2,3-dioxygenase (IDO).

Transcriptional regulation of the interferon-gamma-inducible tryptophanyl-tRNA synthetase includes alternative splicing.

We have investigated the transcriptional control elements of the human interferon (IFN)-gamma-induced tryptophanyl-tRNA synthetase (hWRS) gene and characterized the transcripts. Transcription leads to a series of mRNAs with different combinations of the first exons. The full-length mRNA codes for a 55-kDa protein (hWRS), but a mRNA lacking exon II is present in almost as high amounts as the full-length transcript. This alternatively spliced mRNA is probably translated into a 48-kDa protein starting from Met48 in exon III. The predicted 48-kDa protein corresponds exactly to an IFN-gamma-inducible protein previously detected by two-dimensional gel electrophoresis. By isolation of genomic clones and construction of plasmids containing hWRS promoter fragments fused to the secreted alkaline phosphatase reporter gene we have mapped a promoter region essential for IFN-mediated gene activation. This region contains IFN-stimulated response elements (ISRE) as well as a Y-box and a gamma-activated sequence (GAS) element. IFN-gamma inducibility of hWRS depends on ongoing protein synthesis, suggesting that so far undescribed transcription factors apart from the latent GAS-binding protein p91 contribute to gene activation. This could be interferon-regulatory factor-1, which binds ISRE elements.

Are the tryptophanyl-tRNA synthetase and the peptide-chain-release factor from higher eukaryotes one and the same protein?

Recently, cDNA clones encoding the bovine (b) [M. Garret, B. Pajot, V. Trézéguet, J. Labouesse, M. Merle, J.-C. Gandar, J.-P. Benedetto, M.-L. Sallafranque, J. Alterio, M. Gueguen, C. Sarger, B. Labouesse and J. Bonnet (1991) Biochemistry 30, 7809-7817] and human (h) [L. Yu. Frolova, M. A. Sudomoina, A. Yu. Grigorieva, O. L. Zinovieva and L. L. Kisselev (1991) Gene 109, 291-296] tryptophanyl-tRNA synthetases (TrpRS) were sequenced; the deduced amino acid sequences exhibit typical structural features of class I aminoacyl-tRNA synthetases [G. Eriani, M. Delarue, O. Poch, J. Gangloff and D. Moras (1990) Nature 237, 203-206] and limited, although significant, similarity with bacterial TrpRS. Independently, it was shown that a major protein whose synthesis is stimulated in human cell cultures by interferon gamma [J. Fleckner, H. H. Rasmussen and J. Justesen (1991) Proc. Natl Acad. Sci. USA 88, 11,520-11,524], and interferons gamma or alpha [B. Y. Rubins, S. L. Anderson, L. Xing, R. J. Powell and W. P. Tate (1991) J. Biol. Chem. 226, 24,245-24,248], exhibits TrpRS activity and an amino acid sequence identical to that of hTrpRS. The amino acid sequences of bTrpRS and hTrpRS are highly similar and are surprisingly very similar to the amino acid sequence deduced from a cloned and sequenced cDNA reported to encode rabbit (r) peptide-chain-release factor (RF) [C. C. Lee, W. J. Craigen, D. M. Muzny, E. Harlow and C. T. Caskey (1990) Proc. Natl Acad. Sci. USA 87, 3508-3512]. This close similarity between mammalian TrpRS and cloned RF is unexpected given the distinct functional properties of these proteins. Consequently, the question arises as to whether the mammalian TrpRS and RF activities reside on identical or very similar polypeptides. Alternatively, one may assume that the cloned rabbit cDNA encodes a protein other than rRF. Several properties (immunochemical, biochemical and physico-chemical) of mammalian TrpRS and RF have been compared. rTrpRS and rRF have distinct thermostability behaviours, and dissimilar chromatographic profiles on phosphocellulose. Both the anti-bTrpRS polyclonal antibodies and the monoclonal antibody Am2 strongly inhibit the bTrpRS and hTrpRS aminoacylation activities, but not the rRF activity. In addition, neither bTrpRS nor hTrpRS exhibit RF activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Human interferon gamma potently induces the synthesis of a 55-kDa protein (gamma 2) highly homologous to rabbit peptide chain release factor and bovine tryptophanyl-tRNA synthetase.

An interferon gamma (IFN-gamma)-inducible protein, gamma 2, was identified by two-dimensional gel electrophoresis of transformed human amnion (AMA) cell proteins. cDNA clones coding for this protein have been isolated and characterized as encoding a polypeptide with a predicted molecular weight of 53,165 and a pI of 6.16. Both values are in good agreement with those observed in two-dimensional gel electrophoresis. The gamma 2 protein is found to be highly induced by IFN-gamma, whereas no induction was seen after addition of IFN-alpha to AMA cells. A gamma 2-specific 2.7-kilobase mRNA was likewise seen to accumulate selectively in response to IFN-gamma in these cells. Comparison of the predicted amino acid sequence of gamma 2 to proteins in GenBank data bases revealed that gamma 2 is highly homologous to rabbit peptide chain release factor [Lee, C. C., Craigen, W. J., Muzny, D. M., Harlow, E. & Caskey, C. T. (1990) Proc. Natl. Acad. Sci. USA 87, 3508-3512] and bovine tryptophanyl-tRNA synthetase [M. Garret, V. Trezeguet, B. Pajot, J. C. Gandar, M. Merle, M. Guegiev, J. P. Benedetto, C. Sarger, J. Alteriot, J. La Bouessec, J. Labouesse, and J. Bonnet (1990), GenBank accession no. X52113]. Amino acid sequence similarities of 94% and 97%, respectively, are found, which in general would indicate that gamma 2 represents the human equivalent to either of these two mammalian genes. Based on these sequence similarities, the current data raise the possibility that tryptophanyl-tRNA charging and peptide chain release are carried out by the same enzyme. The gamma 2 protein is shown to possess tryptophan-dependent aminoacyl-tRNA synthetase activity and thus constitutes an enzymatic activity involved in the biological activity of IFN-gamma.

Interferon stimulates the expression of 2',5'-oligoadenylate synthetase and MHC class I antigens in insulin-producing cells.

The pathogenesis of type 1 diabetes involves autoimmune processes directed against the pancreatic beta-cells. The etiology is not known, but circumstantial evidence suggests a connection between virus infection and development of the disease. Therefore, because the interferon-(IFN) dependent 2',5'-oligoadenylate (2-5A) synthetase system constitutes an important part of the nonspecific immune defense against viral infections, the activity of the enzyme was examined in islets of Langerhans, RIN cells, and GH3 cells. First, the 2-5A synthetase was expressed constitutively in all cell types and, second, all cells were sensitive to stimulation with IFN-alpha. The 2-5A synthetase activity induced by 1,000 U/ml of IFN-alpha increased by 400% in pancreatic islets and by more than 1000% in GH3 and RIN cells. However, the IFN-alpha concentration needed to induce half-maximal 2-5A synthetase activity was nearly the same in the three cell types (i.e., ranging from 59 to 66 U/ml IFN-alpha). The 2-5A synthetase present in islets and RIN cells was highly sensitive to poly (I:C). In pancreatic islets and RIN cells, the 2-5A synthetase enzyme generated dimers and trimers of 2',5'-oligoadenylates. Furthermore, exposure of RIN cells to IFN-alpha showed an increase in MHC class I expression already at 5 U/ml and maximal expression at about 200 U/ml IFN-alpha. The examined endocrine cells express the 2-5A synthetase enzyme as well as MHC class I antigen constitutively, but also by stimulation with IFN in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)