Ute Ungethüm

The stress protein BiP is overexpressed and is a major B and T cell target in rheumatoid arthritis

OBJECTIVE The ubiquitously expressed intracellular protein formerly designated p68 has been identified as autoantigen at both the antibody and the T cell level in rheumatoid arthritis (RA). METHODS We used 2 independent approaches, Edman degradation and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, to characterize p68, and we compared its features with those of the endoplasmic reticulum stress protein BiP. RESULTS In synovial sections from RA patients, BiP was highly overexpressed as compared with control sections. Under in vitro stress conditions, BiP was found to translocate to the nucleus and the cell surface. BiP-specific autoantibodies were present in 63% of 400 RA patients, in 7% of 200 patients with other rheumatic diseases, and in none of the healthy subjects. Thus, BiP-specific autoantibodies represent a new diagnostic marker in RA. Furthermore, we found that BiP-specific T cell reactivity was altered in RA. In healthy individuals and patients with other rheumatic diseases, BiP-reactive T cells were undetectable. In RA, overt T cell reactivity to BiP was observed or could be induced by specifically blocking antigen presentation to potentially regulatory T cells. CONCLUSION Since overexpression of BiP has been shown to decrease the sensitivity of cells to killing by cytotoxic T cells, BiP overexpression and BiP-specific autoimmunity may be involved in the pathogenesis of RA.

Systematic evaluation of the miRNA-ome and its downstream effects on mRNA expression identifies gastric cancer progression.

We investigated the differential expression of Dicer and Drosha, as well as that of microRNA (miRNA), in adjacent normal and tumour samples of patients with gastric cancer. The expression of Dicer and Drosha was studied by immunohistochemistry in 332 gastric cancers and correlated with clinico‐pathological patient characteristics. Differential expression of miRNAs was studied using the Invitrogen NCode™ Multi‐Species miRNA Microarray Probe Set containing 857 mammalian probes in a test set of six primary gastric cancers (three with and three without lymph node metastases). Differential expression was validated by RT‐PCR on an independent validation set of 20 patients with gastric cancer. Dicer and Drosha were differentially expressed in non‐neoplastic and neoplastic gastric tissue. The expression of Drosha correlated with local tumour growth and was a significant independent prognosticator of patient survival. Twenty miRNAs were up‐ and two down‐regulated in gastric carcinoma compared with non‐neoplastic tissue. Six of these miRNAs separated node‐positive from node‐negative gastric cancers, ie miR‐103, miR‐21, miR‐145, miR‐106b, miR‐146a, and miR‐148a. Five miRNAs expressed differentially in node‐positive cancers had conserved binding sites for mRNAs differentially expressed in the same set of tumour samples. Gastric cancer shows a complex derangement of the miRNA‐ome, including Dicer and Drosha. These changes correlate independently with patient prognosis and probably influence local tumour growth and nodal spread. Copyright

Differential expression of cathepsins B and L compared with matrix metalloproteinases and their respective inhibitors in rheumatoid arthritis and osteoarthritis: A parallel investigation by semiquantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry

OBJECTIVE To study the expression of the cysteine proteinases cathepsin B and L and their most potent inhibitor cystatin C in the synovial membrane of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) on both the messenger RNA (mRNA) level and the protein level. METHODS The expression of both cysteine proteinases and cystatin C was investigated in synovial tissue from 15 RA and 11 OA patients and compared with the expression of matrix metalloproteinase 1 (MMP-1; collagenase), MMP-3 (stromelysin), and tissue inhibitor of metalloproteinases 1 (TIMP-1). Expression of mRNA was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction. Protein expression was evaluated by immunohistochemistry. The results were correlated with the histologic evidence of inflammatory activity. RESULTS A significantly more pronounced expression of MMP mRNA was observed in RA synovium compared with OA. In contrast, the mRNA expression of cysteine proteinases, as well as TIMP-1 and cystatin C, did not differ between the patient groups. However, the protein expression of both MMP and cysteine proteinases was significantly more prominent in RA synovial lining compared with OA, whereas cystatin C and TIMP-1 protein were expressed equally. CONCLUSION The data indicate a more pronounced expression of MMP mRNA compared with cysteine proteinases in RA. The higher levels of cathepsin B and L proteins in RA synovial lining cells compared with OA are consistent with previous studies that assert a post-transcriptional up-regulation of these enzymes in RA.

Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Bone morphogenetic proteins (BMPs) have been identified as important morphogens with pleiotropic functions in regulating the development, homeostasis and repair of various tissues. The aim of this study was to characterize the expression of BMPs in synovial tissues under normal and arthritic conditions. Synovial tissue from normal donors (ND) and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were analyzed for BMP expression by using microarray hybridization. Differential expression of BMP-4 and BMP-5 was validated by semiquantitative RT-PCR, in situ hybridization and immunohistochemistry. Activity of arthritis was determined by routine parameters for systemic inflammation, by histological scoring of synovitis and by semiquantitative RT-PCR of IL-1β, TNF-α, stromelysin and collagenase I in synovial tissue. Expression of BMP-4 and BMP-5 mRNA was found to be significantly decreased in synovial tissue of patients with RA in comparison with ND by microarray analysis (p < 0.0083 and p < 0.0091). Validation by PCR confirmed these data in RA (p < 0.002) and also revealed a significant decrease in BMP-4 and BMP-5 expression in OA compared with ND (p < 0.015). Furthermore, histomorphological distribution of both morphogens as determined by in situ hybridization and immunohistochemistry showed a dominance in the lining layer of normal tissues, whereas chronically inflamed tissue from patients with RA revealed BMP expression mainly scattered across deeper layers. In OA, these changes were less pronounced with variable distribution of BMPs in the lining and sublining layer. BMP-4 and BMP-5 are expressed in normal synovial tissue and were found decreased in OA and RA. This may suggest a role of distinct BMPs in joint homeostasis that is disturbed in inflammatory and degenerative joint diseases. In comparison with previous reports, these data underline the complex impact of these factors on homeostasis and remodeling in joint physiology and pathology.

Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway dependent gene signatures in colorectal cancer cells.

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

Oncogenic HRAS suppresses clusterin expression through promoter hypermethylation.

Silencing of gene expression by methylation of CpG islands in regulatory elements is frequently observed in cancer. However, an influence of the most common oncogenic signalling pathways onto DNA methylation has not yet been investigated thoroughly. To address this issue, we identified genes suppressed in HRAS-transformed rat fibroblasts but upregulated after treatment with the demethylating agent 5-Aza-2-deoxycytidine and with the MEK1,2 inhibitor U0126. Analysis of gene expression by microarray and Northern blot analysis revealed the MEK/ERK target genes clusterin, matrix metalloproteinase 2 (Mmp2), peptidylpropyl isomerase C-associated protein, syndecan 4, Timp2 and Thbs1 to be repressed in the HRAS-transformed FE-8 cells in a MEK/ERK- and methylation-dependent manner. Hypermethylation of putative regulatory elements in HRAS-transformed cells as compared to immortalized fibroblasts was detected within a CpG island 14.5 kb upstream of clusterin, within the clusterin promoter and within a CpG island of the Mmp2 promoter by bisulphite sequencing. Furthermore, hypermethylation of the clusterin promoter was observed 10 days after induction of HRAS in immortalized rat fibroblasts and a clear correlation between reduced clusterin expression and hypermethlyation could also be observed in distinct rat tissues. These results suggest that silencing of individual genes by DNA methylation is controlled by oncogenic signalling pathways, yet the mechanisms responsible for initial target gene suppression are variable.

Perinatal asphyxia increases bFGF mRNA levels and DA cell body number in the mesencephalon of rats

The present investigation was undertaken in order to study the long-term effects of perinatal asphyxia on basic fibroblast growth factor (bFGF) gene expression and the number of dopamine nerve cell bodies in the mesencephalon of the rat. Asphyxia was induced during birth for 19-20 min. A 30% increase in the number of tyrosine-hydroxylase immunoreactive (TH-IR) nerve cell bodies (i.e. dopamine-containing neurones) as well as a 50% increase in bFGF gene expression following asphyxia was found in the substantia nigra/ventral tegmental area 4 weeks after birth. The increase in bFGF mRNA levels may underlie the increase found in the number of dopamine cell bodies. The present results indicate that asphyxia during birth can prime the long-term development of the central nervous system.

Vascular CXCR4 Expression – a Novel Antiangiogenic Target in Gastric Cancer?

Background G-protein-coupled receptors (GPCRs) are prime candidates for novel cancer prevention and treatment strategies. We searched for differentially expressed GPCRs in node positive gastric carcinomas. Methodology/Principal Findings Differential expression of GPCRs in three node positive vs. three node negative intestinal type gastric carcinomas was analyzed by gene array technology. The candidate genes CXCL12 and its receptor CXCR4 were validated by real-time reverse-transcription polymerase chain reaction in an independent set of 37 gastric carcinomas. Translation was studied by immunohistochemistry in 347 gastric carcinomas using tissue microarrays as well as in 61 matching lymph node metastases. Protein expression was correlated with clinicopathological patient characteristics and survival. 52 GPCRs and GPCR-related genes were up- or down-regulated in node positive gastric cancer, including CXCL12. Differential expression of CXCL12 was confirmed by RT-PCR and correlated with local tumour growth. CXCL12 immunopositivity was negatively associated with distant metastases and tumour grade. Only 17% of gastric carcinomas showed CXCR4 immunopositive tumour cells, which was associated with higher local tumour extent. 29% of gastric carcinomas showed CXCR4 positive tumour microvessels. Vascular CXCR4 expression was significantly associated with higher local tumour extent as well as higher UICC-stages. When expressing both, CXCL12 in tumour cells and CXCR4 in tumour microvessels, these tumours also were highly significantly associated with higher T- and UICC-stages. Three lymph node metastases revealed vascular CXCR4 expression while tumour cells completely lacked CXCR4 in all cases. The expression of CXCL12 and CXCR4 had no impact on patient survival. Conclusions/Significance Our results substantiate the significance of GPCRs on the biology of gastric carcinomas and provide evidence that the CXCL12-CXCR4 pathway might be a novel promising antiangiogenic target for the treatment of gastric carcinomas.

Expression of apoptosis-related genes in the organ of Corti, modiolus and stria vascularis of newborn rats

Cell death in the inner ear tissues is an important mechanism leading to hearing impairment. Here, using microarrays and real-time RT-PCR we analyzed expression of selected apoptosis-related genes in rats inner ear. We determined the gene expression in tissues freshly isolated from neonatal rats (3-5 days old) and compared it to that of explants cultured for 24 h under normoxic or hypoxic conditions. For the analyses, we used pooled samples of the organ of Corti (OC), modiolus (MOD) and stria vascularis (SV), respectively. We observed region-specific changes in gene expression between the fresh tissues and the normoxic culture. In the OC, expression of the proapoptotic genes caspase-2, caspase-3, caspase-6 and calpain-1 was downregulated. In the MOD, the antioxidative defense SOD-2 and SOD-3 were upregulated. In the SV, caspase-2, caspase-6, calpain-1 and SOD-3 were downregulated and SOD-2 upregulated. We speculate that these changes could reflect survival shift in transcriptome of inner ear explants tissues under in vitro conditions. With the exception of SOD-2, hypoxic culture conditions induced the same changes in gene expression as the normoxic conditions indicating that culture preparation is likely the dominating factor, which modifies the gene expression pattern. We conclude that various culture conditions induce different expression pattern of apoptosis-related genes in the organotypic cochlear cultures, as compared to fresh tissues. This transcriptional pattern may reflect the survival ability of specific tissues and could become a tempting target for a pharmacological intervention in inner ear diseases.

De novo expression of EphA2 in osteosarcoma modulates activation of the mitogenic signalling pathway

Fritsche‐Guenther R, Noske A, Ungethüm U, Kuban R‐J, Schlag PM, Tunn P‐U, Karle J, Krenn V, Dietel M & Sers C (2010) Histopathology57, 836–850