Leukocyte gene expression signatures in antineutrophil cytoplasmic autoantibody and lupus glomerulonephritis.

Leukocytes play a major role in the development and progression of autoimmune diseases. We measured gene expression differences in leukocytes from patients that were antineutrophil cytoplasmic autoantibody (ANCA) positive, patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), and healthy donors to explore potential pathways for clinical intervention. Leukocyte gene expression profiles were determined on Affymetrix U133A/B chips in 88 autoimmune patients, 28 healthy donors, and healthy donor leukocyte cell subtypes that were activated in vitro. Comparison of gene expression in leukocytes identified differentially expressed signature genes that distinguish each donor source. The microarray expression levels for many signature genes correlated with the clinical activity of small vessel vasculitis in the ANCA patients; a result confirmed by quantitative real time-polymerase chain reaction for 16 relevant genes. Comparison with in vitro-activated leukocyte subtypes from healthy donors revealed that the ANCA signature genes were expressed by neutrophils while the SLE signature genes were expressed in activated monocytes and T cells. We have found that leukocyte gene expression data can differentiate patients with RA, SLE, and ANCA-related small vessel vasculitis. Monitoring changes in the expression of specific genes may be a tool to help quantify disease activity during treatment.

Mapping of myeloperoxidase epitopes recognized by MPO-ANCA using human-mouse MPO chimers.

Myeloperoxidase (MPO) is one of the major target antigens of antineutrophil cytoplasmic autoantibodies (ANCA) found in patients with small-vessel vasculitis and pauci-immune necrotizing glomerulonephritis. To date, the target epitopes of MPO-ANCA remain poorly defined. Human MPO-ANCA do not typically bind mouse MPO. We utilized the differences between human and mouse MPO to identify the target regions of MPO-ANCA. We generated five chimeric MPO molecules in which we replaced different segments of the human or mouse molecules with their homologous counterpart from the other species. Of serum samples from 28 patients screened for this study, 43 samples from 14 patients with MPO-ANCA-associated vasculitis were tested against recombinant human and mouse MPO and the panel of chimeric molecules. Sera from 64 and 71% of patients bound to the carboxy-terminus of the heavy chain, in the regions of amino acids 517-667 or 668-745, respectively. No patient serum bound the MPO light chain or the amino-terminus of the heavy chain. All sera bound to only one or two regions of MPO. Although the pattern of MPO-ANCA binding changed over time (4-27 months) in 6 of 10 patients with several serum samples, such changes were infrequent. Other target regions of MPO-ANCA may not have been detected due to conformational differences between the native and recombinant forms of MPO. MPO-ANCA do not target a single epitope, but rather a small number of regions of MPO, primarily in the carboxy-terminus of the heavy chain.

ANCA antigens, proteinase 3 and myeloperoxidase, are not expressed in endothelial cells.

BACKGROUND: One hypothesis for the pathogenesis of vasculitis associated with antineutrophil cytoplasmic autoantibodies (ANCAs) proposes that ANCAs bind to ANCA antigens, such as proteinase 3 (PR3) or myeloperoxidase (MPO), which are produced by endothelial cells and expressed on their surfaces. There are conflicting reports, however, on whether endothelial cells express the ANCA antigen PR3, and there are no reports on endothelial expression of MPO. The aim of this study was to determine the presence or absence of PR3 and MPO mRNA in both venous and arterial endothelial cells, employing standard reverse transcription-polymerase chain reaction (RT-PCR) techniques and also the quantitative and highly specific method, TaqMan PCR. METHODS: RT-PCR (with 3 primer sets) and TaqMan PCR, a method for detecting low copy transcripts, were used to probe for PR3 and MPO transcripts in human endothelial cells from umbilical vein (HUVEC) and artery (HUAEC) and from lung microvascular (HLMVEC). Cells were treated with interferon-gamma (200 units/mL) or tumor necrosis factor-alpha (3 or 10 ng/mL) or both. RESULTS: Transcripts for PR3 and/or MPO were not detected in HUVEC, HUAEC, and HLMVEC by standard RT-PCR. Analyses for PR3 protein confirmed that PR3 is not expressed in HUVEC. HUVEC and HUAEC were negative for PR3 and MPO by TaqMan PCR. CONCLUSIONS: PR3 and MPO are not expressed in HUVEC, HUAEC, or HLMVEC. Endothelial cell presentation of endogenous PR3 and MPO antigens is not involved in the pathogenesis of ANCA-associated vasculitis. Alternative explanations need to be explored to determine the pathogenic effect of ANCAs.

Future molecular approaches to the diagnosis and treatment of glomerular disease.

Current diagnoses and treatment decisions for renal disease are made based upon a combination of clinical and pathological determinations. With the advances in both biochemical and molecular biological techniques, identifying the underlying biochemical and genetic changes that may have initiated and/or contributed to the disease is possible. We describe here technologies that may lead to significant changes in renal disease diagnosis, characterization, treatment, and potentially prevention. For example, differential display techniques and DNA gene chip arrays show the changes in mRNA expression patterns and can potentially identify previously unknown genes and reveal new roles for previously known genes in renal disease. The generation of the single nucleotide polymorphisms (SNP) genomic map will facilitate genetic screening that may identify a gene or combination of genes that produce enhanced disease susceptibility. Combining genomic analysis with epidemiological studies may identify environmental factors that contribute to renal disease onset in genetically susceptible individuals. A number of novel therapies are already on the horizon. These include reagents that abrogate the function of specific cytokines, chemokines, and effector cells. With the list of renal disease genes in hand, their role in renal physiology and pathophysiology can be determined, which should lead to the discovery of pharmacological intervention directed at those genes and their products that play a role in the pathogenesis of renal disease.

Two posttranscriptional pathways that regulate p21(Cip1/Waf1/Sdi1) are identified by HPV16-E6 interaction and correlate with life span and cellular senescence.

The p21((Cip1/Waf1/Sdi1)) protein is a cyclin-dependent kinase inhibitor that is induced in normal human fibroblasts (NHF) following DNA damage, following serum stimulation, and at cellular senescence. Expression of the human papilloma virus 16 E6 oncoprotein in NHF cells results in the loss of the p21 protein, independent of mRNA level under most conditions. The p21 protein levels in NHF-E6 cells remained low following DNA damage or serum stimulation even though mRNA levels increased. In contrast, the p21 protein was transiently induced in NHF-E6 cells at the onset of cellular senescence. Expression of the E6 oncoprotein in transformed cells had no effect on p21 protein levels. This demonstrates that two posttranscriptional pathways regulate expression of p21 protein in NHF cells under different conditions. Disruption of posttranscriptional regulation is correlated with extension of life span, altered cell fate, and transformation.

Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.

Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.

Induction of apoptosis by c-Fos protein.

The role of c-Fos in apoptosis was examined in two Syrian hamster embryo cell lines (sup+I and sup-II) and a human colorectal carcinoma cell line (RKO), using the chimeric Fos-estrogen receptor fusion protein c-FosER. As previously reported, contrasting responses were observed when these two cell lines were placed under growth factor deprivation conditions; sup+I cells were highly susceptible to apoptosis, whereas sup-II cells were resistant. In this report, we show that the activated c-FosER protein induces apoptosis in sup-II preneoplastic cells in serum-free medium, indicating that c-Fos protein can induce apoptotic cell death in these cells. c-Fos-induced apoptosis was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that the c-Fos transcriptional activation activity is not involved. This conclusion was further supported by the observation that overexpression of v-Fos, which is highly proficient in transcriptional activation but deficient in the transcriptional repression activity associated with c-Fos, did not induce apoptosis. Constitutively expressed Bcl-2 delayed the onset of low-serum-induced apoptosis in sup+I cells and enhanced survival in sup-II cells. Further, coexpression of Bcl-2 and c-FosER in sup+I or sup-II cells protected the cells from c-FosER-induced apoptosis. The possibility that c-FosER-induced apoptosis requires a p53 function was examined. Colorectal carcinoma RKOp53+/+ cells, which do not normally undergo apoptosis in serum-free medium, showed apoptotic DNA fragmentation upon expression and activation of c-FosER. Further, when the wild-type p53 protein was diminished in the RKO cells by infection with the papillomavirus E6 gene, subsequent c-FosER-induced apoptosis was blocked. The data suggest that c-Fos protein plays a causal role in the activation of apoptosis in a p53-dependent manner. This activity does not require new protein synthesis and is blocked by overexpression of Bcl-2 protein.

p21CIP1 is not required for the early G2 checkpoint response to ionizing radiation.

We have previously reported that the immediate G2 checkpoint delay of normal human fibroblasts in response to ionizing radiation is correlated with inhibition of p34CDC2/cyclin B kinase activity. Here, we observed increased amounts of the cyclin-dependent protein kinase inhibitor p21CIP1 associated with p34CDC2/cyclin B protein complexes from irradiated normal human fibroblasts. Since wild-type p53 function is not required for the early G2 checkpoint response to ionizing radiation, we investigated whether a p53-independent induction of p21CIP1 was required for the G2 checkpoint. Early passage human fibroblasts expressing the E6 oncoprotein of human papilloma virus-type 16 (NHF4 E6) were analyzed. It has been demonstrated earlier than inactivation of wild-type p53 function in these cells by E6 protein does not alter their intact early G2 checkpoint response to gamma-rays. p21CIP1 was found to be undetectable in p34CDC2/cyclin B protein complexes and in total extracts from the E6-expressing cells, with or without exposure to ionizing radiation. These data indicate that p21CIP1 is not required for the immediate G2 checkpoint response and is not induced by a p53-independent pathway in G2 phase following exposure to gamma-rays.

Distinct mechanisms for the activation of the RSK kinases/MAP2 kinase/pp90rsk and pp70-S6 kinase signaling systems are indicated by inhibition of protein synthesis.

Previous studies demonstrated that addition of protein synthesis inhibitors to quiescent cells resulted in the stimulation of S6 kinase activity. The present characterization of several growth factor- and oncogene-regulated protein-serine/threonine kinases demonstrated that pp70-S6 protein kinase and not pp90rsk, RSK kinase, or MAP2 kinase activities were rapidly stimulated. Dose-response experiments revealed a close correlation between the extent of protein synthesis inhibition and the level of activation of pp70-S6 kinase activity. Analysis of S6 phosphorylation suggests that activation of pp90rsk S6 phosphotransferase activity, whose Xenopus homologues appear to be responsible for S6 phosphorylation during oocyte maturation, may participate in, but is not essential for, the increase in S6 phosphorylation observed in growth-stimulated somatic animal cells. These studies provide additional evidence for the existence of two distinct, independently regulated protein phosphorylation cascades activated in the early G1 phase of the cell cycle.

Two distinct enzymes contribute to biphasic S6 phosphorylation in serum-stimulated chicken embryo fibroblasts.

Serum stimulation of quiescent chicken embryo fibroblasts resulted in a time-dependent, biphasic activation of S6 kinase activity. Chromatographic fractionation of serum-stimulated cell lysates resolved two distinct S6 kinase activities. Anti-Xenopus S6 kinase II antiserum immunoprecipitated a 90,000-Mr S6 kinase but did not cross-react with a smaller, 65,000-Mr S6 kinase. Phosphopeptide analysis confirmed that the 90,000- and 65,000-Mr proteins were structurally unrelated and established that the 65,000-Mr protein isolated by the current protocol was the same serum-stimulated chicken embryo fibroblast S6 kinase as that previously characterized (J. Blenis, C. J. Kuo, and R. L. Erikson, J. Biol. Chem. 262:14373-14376, 1987). These results demonstrate the contribution of two distinct S6 kinases to total serum-stimulated ribosomal protein S6 phosphorylation.

Identification of mitogen-responsive ribosomal protein S6 kinase pp90rsk, a homolog of Xenopus S6 kinase II, in chicken embryo fibroblasts.

Antiserum raised against recombinant Xenopus ribosomal protein S6 kinase (rsk) was used to identify a 90,000-Mr ribosomal S6 kinase, pp90rsk, in chicken embryo fibroblasts. Adding serum to cells stimulated the phosphorylation of pp90rsk on serine and threonine residues and increased the activity of S6 kinase measured in immune complex assays. Xenopus S6 kinase II and chicken embryo fibroblast pp90rsk had nearly identical phosphopeptide maps.

Sequence and expression of chicken and mouse rsk: homologs of Xenopus laevis ribosomal S6 kinase.

We have previously reported the isolation of cDNAs encoding two closely related Xenopus ribosomal S6 kinases, S6KII alpha and -beta (S. W. Jones, E. Erikson, J. Blenis, J. L. Maller, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 85:3377-3381, 1988). We report here the molecular cloning of one chicken and two mouse homologs of the Xenopus laevis cDNAs. As described for the Xenopus proteins, these cDNAs were found to predict polypeptides that contain two distinct kinase domains, of which one is most closely related to the catalytic subunit of cyclic AMP-dependent protein kinase and the other is most closely related to the catalytic subunit of phosphorylase b kinase. The three predicted proteins were more than 79% identical to the Xenopus S6KII alpha protein. The chicken and one of the mouse cDNAs were, respectively, 3.7 and 3.1 kilobase pairs in length, predicted proteins of 752 and 724 amino acids with molecular weights of 84.4 and 81.6 kilodaltons, and hybridized to mRNAs in fibroblasts and tissues of approximately 3.6 and 3.4 kilobases (kb). The second mouse cDNA was approximately 6.1 kilobase pairs and was not full length but predicted the C-terminal 633 amino acids of a protein that is similar to the C-terminal portion of Xenopus S6KII alpha. This clone hybridized to mRNA transcripts of 7.6 and 3.4 kb. In vitro transcription and translation of the chicken and the mouse cDNAs that predict complete proteins produced major products with apparent molecular weights of 96 and 84 kilodaltons. Analysis of mRNA levels in chicken tissues showed significant quantities of the 3.6-kb transcript in small and large intestine, spleen, and bursa. Both mouse cDNA were similarly expressed at significant levels in intestine, thymus, and lung; however, the 7.6-kb mRNA was differentially and more highly expressed in heart and brain. The two mouse cDNAs represent two different S6 kinase genes, as shown by comparison of their protein sequences, mRNA transcript sizes, genomic organizations, and nucleic acid sequences. We propose that this family of genes be named rsk, for ribosomal S6 kinase.