References of "Behrmann, Iris 50000694"
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See detailBiological responses to PDGF-BB versus PDGF-DD in human mesangial cells
van Roeyen, C. R.; Ostendorf, T.; Denecke, B. et al

in Kidney International (2006), 69(8), 1393-402

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more ... [more ▼]

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor. [less ▲]

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See detailThree dileucine-like motifs within the interbox1/2 region of the human oncostatin M receptor prevent efficient surface expression in the absence of an associated Janus kinase
Radtke, S.; Jörissen, A.; de Leur, H. S. et al

in Journal of Biological Chemistry (2006), 281(7), 4024-34

The oncostatin M receptor (OSMR) is part of receptor complexes for oncostatin M and interleukin-31. Signaling events are triggered by Jaks (Janus kinases) that constitutively bind to membrane-proximal ... [more ▼]

The oncostatin M receptor (OSMR) is part of receptor complexes for oncostatin M and interleukin-31. Signaling events are triggered by Jaks (Janus kinases) that constitutively bind to membrane-proximal receptor regions. Besides their established role in signaling, Jaks are involved in the regulation of the surface expression of several cytokine receptors. Here, we analyzed the structural requirements within the human OSMR that underlie its limited surface expression in the absence of associated Jaks. We identified three dileucine-like motifs within the Jak-binding region of the OSMR that control receptor surface and overall expression. A receptor mutant in which all three motifs were mutated to alanine displayed markedly increased surface expression. Although the surface half-life of this mutant was increased compared with that of the wild-type receptor, no difference in the internalization rate was detectable, implying that these receptors differ in their post-endocytic fate. The protein stability of the wild-type receptor was markedly lower than that of mutant receptors, but could be strongly increased in the presence of the lysosomal inhibitor chloroquine. Our data are consistent with the dileucine motifs being involved in destabilization of receptors devoid of associated Jaks as part of a quality control ensuring signaling competence of OSMRs. [less ▲]

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See detailPromoter-hypermethylation is causing functional relevant downregulation of methylthioadenosine phosphorylase (MTAP) expression in hepatocellular carcinoma
Hellerbrand, C.; Mühlbauer, M.; Wallner, S. et al

in Carcinogenesis (2005), 27(1), 64-72

The methylthioadenosine phosphorylase (MTAP) gene is localized in the chromosomal region 9p21. Here, frequently homozygous deletions occur in several kinds of cancer associated with the loss of tumour ... [more ▼]

The methylthioadenosine phosphorylase (MTAP) gene is localized in the chromosomal region 9p21. Here, frequently homozygous deletions occur in several kinds of cancer associated with the loss of tumour suppressor genes as p16 and p15. The aim of this study was to analyse MTAP expression in hepatocellular carcinoma (HCC) and to get an insight into the regulation and functional role of MTAP in hepatocancerogenesis. Compared with primary human hepatocytes MTAP expression was markedly downregulated in three different HCC cell lines as determined by real-time PCR and western blotting. This was not due to genomic losses or mutations but to promoter-hypermethylation. Reduced MTAP-expression was confirmed in vivo in HCC compared with non-cancerous liver tissue on both mRNA and protein levels. To study the functional relevance of the downregulated MTAP expression in HCC, MTAP expression was re-induced in HCC cell lines by stable transfection. In these MTAP re-expressing cell clones the invasive potential was strongly reduced, whereas no effects on cell proliferation were observed in comparison with mock transfected cell clones. Furthermore, in MTAP re-expressing cells interferon (IFN)-alpha and IFN-gamma induced a significantly stronger inhibition of cell proliferation than in mock transfected cells. In conclusion, our results suggest a functional role of MTAP inactivation in HCC development and invasiveness. Furthermore, in the light of a recent report revealing an association between MTAP activity and IFN sensitivity, our findings may have clinical significance for therapeutic strategies. [less ▲]

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See detailThe Jak1 SH2 domain does not fulfill a classical SH2 function in Jak/STAT signaling but plays a structural role for receptor interaction and up-regulation of receptor surface expression
Radtke, S.; Haan, Serge UL; Jörissen, A. et al

in Journal of Biological Chemistry (2005), 280(27), 25760-8

The presence of a Src homology 2 (SH2) domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study ... [more ▼]

The presence of a Src homology 2 (SH2) domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study to determine the function of the Jak1 SH2 domain. We investigated the functionality of the Jak1 SH2 domain by stably reconstituting Jak1-defective human fibrosarcoma cells U4C with endogenous amounts of Jak1 in which the crucial arginine residue Arg466 within the SH2 domain has been replaced by lysine. This mutant still binds to the receptor subunits gp130 and OSMR. Moreover, the SH2 R466K mutation does not affect the subcellular distribution of Jak1 as assessed by cell fractionation and confocal microscopy of cells expressing endogenous levels of non-tagged or a yellow fluorescent protein (YFP)-tagged Jak1-R466K, respectively. Likewise, the signaling capacity of Jak1 was not affected by this point mutation. However, we found that the SH2 domain is structurally important for cytokine receptor binding and surface expression of the OSMR. [less ▲]

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See detailSTAT5 contributes to interferon resistance of melanoma cells
Wellbrock, C.; Weisser, C.; Hassel, J. C. et al

in Current Biology (2005), 15(18), 1629-39

BACKGROUND: Malignant melanoma is a highly aggressive neoplastic disease whose incidence is increasing rapidly. In recent years, the use of interferon alpha (IFNalpha) has become the most established ... [more ▼]

BACKGROUND: Malignant melanoma is a highly aggressive neoplastic disease whose incidence is increasing rapidly. In recent years, the use of interferon alpha (IFNalpha) has become the most established adjuvant immunotherapy for melanoma of advanced stage. IFNalpha is a potent inhibitor of melanoma cell proliferation, and the signal transducer and activator of transcription STAT1 is crucial for its antiproliferative action. Although advanced melanomas clinically resistant to IFNalpha are frequently characterized by inefficient STAT1 signaling, the mechanisms underlying advanced-stage interferon resistance are poorly understood. RESULTS: Here, we demonstrate that IFNalpha activates STAT5 in melanoma cells and that in IFNalpha-resistant cells STAT5 is overexpressed. Significantly, the knockdown of STAT5 in interferon-resistant melanoma cells restored the growth-inhibitory response to IFNalpha. When STAT5 was overexpressed in IFNalpha-sensitive cells, it counteracted interferon-induced growth inhibition. The overexpressed STAT5 diminished IFNalpha-triggered STAT1 activation, most evidently through upregulation of the inhibitor of cytokine-signaling CIS. CONCLUSIONS: Our data demonstrate that overexpression and activation of STAT5 enable melanoma cells to overcome cytokine-mediated antiproliferative signaling. Thus, overexpression of STAT5 can counteract IFNalpha signaling in melanoma cells, and this finally can result in cytokine-resistant and progressively growing tumor cells. These findings have significant implications for the clinical failure of IFNalpha therapy of advanced melanoma because they demonstrate that IFNalpha induces the activation of STAT5 in melanoma cells, and in STAT5-overexpressing cells, this contributes to IFNalpha resistance. [less ▲]

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See detailMultiple reasons for an inefficient STAT1 response upon IL-6-type cytokine stimulation
Haan, Serge UL; Keller, J. F.; Behrmann, Iris UL et al

in Cellular Signalling (2005), 17(12), 1542-50

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and ... [more ▼]

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and fertility. We found that IL-6-type cytokine stimulation of cell lines and primary human macrophages results in a different distribution of the DNA-binding competent STAT dimer species in the cytosol and nucleus as demonstrated by electrophoretic mobility shift assays. In the absence of detergent, STAT3/STAT3, STAT1/STAT3 were the predominant species in the cytoplasm while STAT3/STAT3 was predominant in the nucleus. However, in detergent containing total cellular lysates and nuclear fractions prepared with detergent containing buffers, the STAT1/STAT1 homodimer was as prominent or even more prominent than STAT3/STAT3 and STAT1/STAT3. We were interested in the cause of this discrepancy since STAT1-regulated genes have not been described to be expressed upon IL-6-type cytokine stimulation. In addition to the more transient STAT1 activation, IL-6-type cytokines such as IL-6 and OSM lead to a much less efficient STAT1 activation compared to the potent STAT1 activators IFNgamma and IFNalpha. Studies with STAT1-deficient cells revealed that STAT1 activation does not seem to be an important competitive process to STAT3 activation arguing again for a very inefficient STAT1 activation upon IL-6-type cytokine stimulation. We also describe that pY-STAT3 is much more efficiently shuttled into the nucleus than pY-STAT1. [less ▲]

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See detailAre STATS arginine-methylated?
Komyod, W.; Bauer, U. M.; Heinrich, P. C. et al

in Journal of Biological Chemistry (2005), 280(23), 21700-5

Transcription factors of the STAT (signal transducer and activator of transcription) family are important in signal transduction of cytokines. They are subject to post-translational modification by ... [more ▼]

Transcription factors of the STAT (signal transducer and activator of transcription) family are important in signal transduction of cytokines. They are subject to post-translational modification by phosphorylation on tyrosine and serine residues. Recent evidence suggested that STATs are methylated on a conserved arginine residue within the N-terminal region. STAT arginine methylation has been described to be important for STAT function and loss of arginine methylation was discussed to be involved in interferon resistance of cancer cells. Here we provide several independent lines of evidence indicating that the issue of arginine methylation of STATs has to be reassessed. First, we show that treatment of melanoma and fibrosarcoma cells with inhibitors used to suppress methylation (N-methyl-2-deoxyadenosine, adenosine, dl-homocysteine) had profound and rapid effects on phosphorylation of STAT1 and STAT3 but also on p38 and Erk signaling cascades which are known to cross-talk with the Jak/STAT pathway. Second, we show that anti-methylarginine antibodies did not precipitate specifically STAT1 or STAT3. Third, we show that mutation of Arg(31) to Lys led to destabilization of STAT1 and STAT3, implicating an important structural role of Arg(31). Finally, purified catalytically active protein arginine methyltransferases (PRMT1, -2, -3, -4, and -6) did not methylate STAT proteins, and cotransfection with PRMT1 did not affect STAT1-controlled reporter gene activity. Taken together, our data suggest the absence of arginine methylation of STAT1 and STAT3. [less ▲]

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See detailJanus kinase (Jak) subcellular localization revisited: the exclusive membrane localization of endogenous Janus kinase 1 by cytokine receptor interaction uncovers the Jak.receptor complex to be equivalent to a receptor tyrosine kinase
Behrmann, Iris UL; Smyczek, Tanja; Heinrich, Peter C. et al

in Journal of Biological Chemistry (2004), 279(34), 35486-93

The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal ... [more ▼]

The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor.Janus kinase complexes can be regarded as receptor tyrosine kinases. [less ▲]

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See detailInterferon-gamma-mediated growth regulation of melanoma cells: involvement of STAT1-dependent and STAT1-independent signals
Kortylewski, M.; Komyod, W.; Kauffmann, M. E. et al

in Journal of Investigative Dermatology (2004), 122(2), 414-22

Interferon-gamma, a known inhibitor of tumor cell growth, has been used in several protocols for the treatment of melanoma. We have studied the molecular events underlying interferon-gamma-induced G0/G1 ... [more ▼]

Interferon-gamma, a known inhibitor of tumor cell growth, has been used in several protocols for the treatment of melanoma. We have studied the molecular events underlying interferon-gamma-induced G0/G1 arrest in four metastatic melanoma cell lines with different responsiveness to interferon-gamma. The growth arrest did not result from enhanced expression of cyclin-dependent kinase inhibitors p21 and p27. Instead, it correlated with downregulation of cyclin E and cyclin A and inhibition of their associated kinase activities. We show that interferon-gamma-induced growth inhibition could be abrogated by overexpression of dominant negative STAT1 (signal transducer and activator of transcription 1) in the melanoma cell line A375, suggesting that STAT1 plays a crucial part for the anti-proliferative effect. Erythropoietin stimulation of a chimeric receptor led to a concentration-dependent STAT1 activation and concomitant growth arrest when it contained the STAT recruitment motif Y440 of the interferon-gamma receptor 1. In contrast, dose-response studies for interferon-gamma revealed a discrepancy between levels of STAT1 activation and the extent of growth inhibition; whereas STAT1 was activated by low doses of interferon-gamma (10 U per mL), growth inhibitory effects were only visible with 100-fold higher concentrations. Our results suggest the presence of additional signals emanating from the interferon-gamma receptor, which may counteract the anti-proliferative function of STAT1. [less ▲]

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See detailThe role of the inhibitors of interleukin-6 signal transduction SHP2 and SOCS3 for desensitization of interleukin-6 signalling
Fischer, P.; Lehmann, U.; Sobota, R. M. et al

in Biochemical Journal (2004), 378(Pt 2), 449-60

The immediate early response of cells treated with IL-6 (interleukin-6) is the activation of the signal transducer and activator of transcription (STAT)3. The Src homology domain 2 (SH2)-containing ... [more ▼]

The immediate early response of cells treated with IL-6 (interleukin-6) is the activation of the signal transducer and activator of transcription (STAT)3. The Src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP2 and the feedback inhibitor SOCS3 (suppressor of cytokine signalling) are potent inhibitors of IL-6 signal transduction. Impaired function of SOCS3 or SHP2 leads to enhanced and prolonged IL-6 signalling. The inhibitory function of both proteins depends on their recruitment to the tyrosine motif 759 within glycoprotein gp130. In contrast to inactivation, desensitization of signal transduction is regarded as impaired responsiveness due to prestimulation. Usually, after activation the sensing receptor becomes inactivated by modifications such as phosphorylation, internalization or degradation. We designed an experimental approach which allows discrimination between desensitization and inactivation of IL-6 signal transduction. We observed that pre-stimulation with IL-6 renders cells less sensitive to further stimulation with IL-6. After several hours, the cells become sensitive again. We show that not only signal transduction through previously activated receptors is affected by desensitization but signalling through receptors which were not targeted by the first stimulation was also attenuated ( trans -desensitization). Interestingly, in contrast to inhibition, desensitization does not depend on the presence of functional SHP2. Furthermore, cells lacking SOCS3 show constitutive STAT3 activation which is not affected by pre-stimulation with IL-6. All these observations suggest that desensitization and inhibition of signalling are mechanistically distinct. [less ▲]

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See detailPrinciples of interleukin (IL)-6-type cytokine signalling and its regulation
Heinrich, P. C.; Behrmann, Iris UL; Haan, Serge UL et al

in Biochemical Journal (2003), 374(Pt 1), 1-20

The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important ... [more ▼]

The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important family of mediators involved in the regulation of the acute-phase response to injury and infection. Besides their functions in inflammation and the immune response, these cytokines play also a crucial role in haematopoiesis, liver and neuronal regeneration, embryonal development and fertility. Dysregulation of IL-6-type cytokine signalling contributes to the onset and maintenance of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, multiple sclerosis and various types of cancer (e.g. multiple myeloma and prostate cancer). IL-6-type cytokines exert their action via the signal transducers gp (glycoprotein) 130, LIF receptor and OSM receptor leading to the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) cascades. This review focuses on recent progress in the understanding of the molecular mechanisms of IL-6-type cytokine signal transduction. Emphasis is put on the termination and modulation of the JAK/STAT signalling pathway mediated by tyrosine phosphatases, the SOCS (suppressor of cytokine signalling) feedback inhibitors and PIAS (protein inhibitor of activated STAT) proteins. Also the cross-talk between the JAK/STAT pathway with other signalling cascades is discussed. [less ▲]

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See detailAkt modulates STAT3-mediated gene expression through a FKHR (FOXO1a)-dependent mechanism
Kortylewski, M.; Feld, F.; Krüger, K. D. et al

in Journal of Biological Chemistry (2003), 278(7), 5242-9

The phosphatidylinositol 3-kinase/Akt pathway plays an important role in the signaling of insulin and other growth factors, which reportedly attenuate the interleukin-6 (IL-6)-mediated stimulation of ... [more ▼]

The phosphatidylinositol 3-kinase/Akt pathway plays an important role in the signaling of insulin and other growth factors, which reportedly attenuate the interleukin-6 (IL-6)-mediated stimulation of acute phase plasma protein genes. We investigated the effect of the protein kinase Akt on IL-6-mediated transcriptional activation. The transient expression of constitutively active Akt inhibited the IL-6-dependent activity of the alpha(2)-macroglobulin promoter in HepG2 cells, whereas expression of an inactive mutant of phosphatidylinositol-dependent kinase 1 had the opposite effect. Since Akt is known to regulate gene expression through inactivation of the transcription factor FKHR (forkhead in rhabdomyosarcoma), we examined the effect of FKHR on STAT3-mediated transcriptional regulation. Indeed, the overexpression of FKHR specifically enhanced the activity of STAT3-dependent promoters but not that of a STAT5-responsive promoter. The effect of FKHR required the presence of functional STAT3 and was abrogated by the expression of dominant negative STAT3 mutants. Furthermore, FKHR and STAT3 were shown to coimmunoprecipitate and to colocalize in the nuclear regions of IL-6-treated HepG2 cells. Our results indicate that FKHR can modulate the IL-6-induced transcriptional activity by acting as a coactivator of STAT3. [less ▲]

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See detailCharacterization of methylthioadenosin phosphorylase (MTAP) expression in malignant melanoma
Behrmann, Iris UL; Wallner, S.; Komyod, W. et al

in American Journal of Pathology (2003), 163(2), 683-90

Homozygous deletions of human chromosomal region 9p21 occur frequently in malignant melanoma and are associated with the loss of the tumor suppressor genes p16(INK4a) and p15(INK4b). In the same ... [more ▼]

Homozygous deletions of human chromosomal region 9p21 occur frequently in malignant melanoma and are associated with the loss of the tumor suppressor genes p16(INK4a) and p15(INK4b). In the same chromosomal region the methylthioadenosine phosphorylase (MTAP) gene is localized and therefore may also serve as a tumor suppressor gene. The aim of this study was to analyze MTAP mutations and expression patterns in malignant melanomas. To examine the MTAP gene and expression of MTAP protein we screened 9 human melanoma cell lines and primary human melanocytes by reverse transcriptase-polymerase chain reaction, sequencing, and immunoblotting. Analyzing the melanoma cell lines we found significant down-regulation of MTAP mRNA expression. In only one cell line, HTZ19d, this was due to homozygous deletion of exon 2 to 8 whereas in the other cell lines promoter hypermethylation was detected. MTAP expression was further analyzed in vivo by immunohistochemical staining of 38 tissue samples of benign melanocytic nevi, melanomas, and melanoma metastases. In summary, we demonstrate significant inverse correlation between MTAP protein expression and progression of melanocytic tumors as the amount of MTAP protein staining decreases from benign melanocytic nevi to metastatic melanomas. Our results suggest an important role of MTAP inactivation in the development of melanomas. This finding may be of great clinical significance because recently an association between MTAP activity and interferon sensitivity has been suggested. [less ▲]

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See detailLong term association of the cytokine receptor gp130 and the Janus kinase Jak1 revealed by FRAP analysis
Giese, B.; Au-Yeung, C. K.; Herrmann, A. et al

in Journal of Biological Chemistry (2003), 278(40), 39205-13

Signal transduction through cytokine receptors is mediated mainly by non-covalently associated Jak tyrosine kinases. By confocal microscopy, the cytokine receptor gp130 and Jak1, fused with either yellow ... [more ▼]

Signal transduction through cytokine receptors is mediated mainly by non-covalently associated Jak tyrosine kinases. By confocal microscopy, the cytokine receptor gp130 and Jak1, fused with either yellow (YFP) or cyan (CFP) fluorescent protein, were found to be colocalized predominantly at intracellular vesicular structures and at the plasma membrane. Quantitative fluorescence recovery after photobleaching (FRAP) analysis at the plasma membrane revealed equal mobilities for gp130-YFP and Jak1-YFP. Thus, Jak1-YFP diffuses like a transmembrane protein indicating that membrane-bound Jak1 does not exchange rapidly with cytosolic Jaks. Applying a novel dual-color FRAP approach we found that immobilization of gp130-CFP by a pair of monoclonal antibodies led to a corresponding immobilization of co-transfected Jak1-YFP. We conclude from these findings that Jak1, once bound to a gp130 molecule, does not exchange between different receptors at the plasma membrane neither via the cytoplasmic compartment nor via a membrane-associated state. [less ▲]

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See detailNovel role of Janus kinase 1 in the regulation of oncostatin M receptor surface expression
Radtke, S.; Hermanns, H. M.; Haan, Claude UL et al

in Journal of Biological Chemistry (2002), 277(13), 11297-305

The oncostatin M receptor (OSMR) is part of a heterodimeric receptor complex that mediates signal transduction of the pleiotropic cytokine OSM via a signaling pathway involving Janus kinases (Jaks) and ... [more ▼]

The oncostatin M receptor (OSMR) is part of a heterodimeric receptor complex that mediates signal transduction of the pleiotropic cytokine OSM via a signaling pathway involving Janus kinases (Jaks) and transcription factors of the signal transducers and activators of transcription (STAT) family. Upon heterologous expression of the OSMR in several cell lines, we observed that its surface expression was significantly enhanced by coexpression of the Janus kinases Jak1, Jak2, and Tyk2 but not Jak3. Chimeric receptors consisting of the extracellular region of the interleukin-5 receptor beta chain and the transmembrane and intracellular part of the OSMR were similarly up-regulated on the plasma membrane when Jak1 was coexpressed. The overall expression level of these constructs did not change significantly, but Jak1 coexpression increased the amount of endoglycosidase H-resistant, fully processed OSMR chimeras. Using mutated receptor and Jak1 constructs, we were able to demonstrate that association of Jak1 with the membrane proximal region of the receptor, but not its kinase activity, is necessary for this effect. Moreover, deletion of the OSMR box1/2 region also resulted in an improved surface expression indicating that this region may contain a signal preventing efficient receptor surface expression in the absence of associated Jaks. Finally we demonstrate that in Jak1-deficient cells, the endogenous OSMR is significantly down-regulated, an effect that can be reversed by transient expression of Jak1 in these cells. [less ▲]

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See detailOrientational constraints of the gp130 intracellular juxtamembrane domain for signaling
Greiser, J. S.; Stross, C.; Heinrich, P. C. et al

in Journal of Biological Chemistry (2002), 277(30), 26959-65

The glycoprotein 130 (gp130) is the common signal transducing receptor chain of the interleukin-6 family of cytokines. Here we investigated the requirements for transfer of the information given by ligand ... [more ▼]

The glycoprotein 130 (gp130) is the common signal transducing receptor chain of the interleukin-6 family of cytokines. Here we investigated the requirements for transfer of the information given by ligand binding to the cytoplasmic domain of gp130. It is demonstrated that the box 1/2 region has to be located membrane-proximally in order to bind and activate Janus kinases. To test the possible requirement of an alpha-helical orientation, we inserted 1-4 alanine residues into this juxtamembrane intracellular region. The insertion of one alanine results in a strongly reduced activation of STAT1 and STAT3, whereas insertion of three alanine residues leads to a stronger STAT activation. These results suggest that gp130-mediated activation of STATs is sensitive to rotational changes around the receptor axis perpendicular to the membrane. Surprisingly, insertion of 1, 2, 3, or 4 alanine residues into this juxtamembrane region leads to successive impairment but not abolishment of Janus kinase and receptor phosphorylation, supporting the finding of sensitivity of Janus kinases toward changes in distance of box 1/2 from the plasma membrane. We suggest a new model concerning the gp130 activation mode in which the relative orientation of the cytoplasmic regions seems to be critical for further signal transduction. [less ▲]

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See detailIdentification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers
Bahrenberg, G.; Behrmann, Iris UL; Barthel, A. et al

in Molecular Endocrinology (2002), 16(4), 859-72

Two predominant splice variants of the leptin receptor (LEPR) are coexpressed in leptin-responsive tissues: the long form, LEPRb, characterized as the signal-transducing receptor, and the signaling ... [more ▼]

Two predominant splice variants of the leptin receptor (LEPR) are coexpressed in leptin-responsive tissues: the long form, LEPRb, characterized as the signal-transducing receptor, and the signaling-defective short form, LEPRa. It is unknown whether heterodimers of these isoforms are capable of signal transduction via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. To address this question, chimeric receptors were constructed consisting of the transmembrane and intracellular parts of LEPRb and LEPRa fused with the extracellular domains of either the alpha- or beta-subunit of the IL-5 receptor. This strategy allows the directed heterodimerization of different LEPR cytoplasmic tails and excludes homodimerization. In COS-7 and HEPG2 cells, chimeric receptor heterodimers of LEPRa and LEPRb failed to activate the JAK/STAT pathway, whereas receptor dimers of LEPRb gave rise to the expected ligand-dependent activation of JAK2, phosphorylation of STAT3, and STAT3-dependent promoter activity. Markedly lower amounts of JAK2 were found to be associated with immunoprecipitated LEPRa chimeras than with LEPRb chimeras. Analysis of a series of deletion constructs indicated that a segment of 15 amino acids in addition to the 29 amino acids common to LEPRa and LEPRb was required for partial restoration of JAK/STAT activation. Site-directed mutagenesis of the critical sequence indicated that two hydrophobic residues (Leu896, Phe897) not present in LEPRa were indispensable for receptor signaling. These findings show that LEPRa/LEPRb heterodimers cannot activate STAT3 and identify sequence elements within the LEPR that are critical for the activation of JAK2 and STAT3. [less ▲]

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See detailThe matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist
Roeb, E.; Schleinkofer, K.; Kernebeck, T. et al

in Journal of Biological Chemistry (2002), 277(52), 50326-32

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin ... [more ▼]

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy. [less ▲]

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See detailSHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signaling through gp130
Lehmann, U.; Schmitz, J.; Weissenbach, M. et al

in Journal of Biological Chemistry (2002), 278(1), 661-71

Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being ... [more ▼]

Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130. [less ▲]

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Peer Reviewed
See detailNeutralizing monoclonal antibodies can potentiate IL-5 signaling
Zabeau, L.; Van der Heyden, J.; Broekaert, D. et al

in European Journal of Immunology (2001), 31(4), 1087-97

IL-5 is a major determinant in the survival, differentiation and effector-functions of eosinophils. It mediates its effect upon binding and activation of a membrane bound receptor (R), composed of a ... [more ▼]

IL-5 is a major determinant in the survival, differentiation and effector-functions of eosinophils. It mediates its effect upon binding and activation of a membrane bound receptor (R), composed of a ligand-specific alpha-chain and a beta-chain, shared with the receptors for IL-3 and granulocyte-macrophage colony-stimulating factor. We have generated and mapped the epitopes of three monoclonal antibodies (mAb) directed against this cytokine: the strong neutralizing mAb 5A5 and 1E1, and the very weak neutralizing mAb H30. We found that H30 as well as 5A5 can increase proliferation above the level induced by human (h)IL-5 alone, in a JAK-2-dependent manner, and at every sub-optimal hIL-5 concentration analyzed. This effect is dependent on mAb-mediated cross-linking of IL-5R complexes, and is only observed on cell lines expressing a hybrid human/mouse IL-5Ralpha-chain. We discuss these findings in view of the stoichiometric and topological requirements for an activated IL-5R. Since humanized anti-IL-5 mAb are currently in clinical testing, our findings imply that such mAb should be carefully evaluated for their potentiating effects. [less ▲]

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