Jörn Krätzschmar

Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1

Glucocorticoids inhibit the proinflammatory activities of transcription factors such as AP‐1 and NF‐κB as well as that of diverse cellular signaling molecules. One of these signaling molecules is the extracellular signal‐regulated kinase (Erk‐1/2) that controls the release of allergic mediators and the induction of proinflammatory cytokine gene expression in mast cells. The mechanism of inhibition of Erk‐1/2 activity by glucocorticoids is unknown. Here we report a novel dual action of glucocorticoids for this inhibition. Glucocorticoids increase the expression of the MAP kinase phosphatase‐1 (MKP‐1) gene at the promoter level, and attenuate proteasomal degradation of MKP‐1, which we report to be triggered by activation of mast cells. Both induction of MKP‐1 expression and inhibition of its degradation are necessary for glucocorticoid‐mediated inhibition of Erk‐1/2 activation. In NIH‐3T3 fibroblasts, although glucocorticoids up‐regulate the MKP‐1 level, they do not attenuate the proteasomal degradation of this protein and consequently they are unable to inhibit Erk‐1/2 activity. These results identify MKP‐1 as essential for glucocorticoid‐mediated control of Erk‐1/2 activation and unravel a novel regulatory mechanism for this anti‐inflammatory drug.

The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundas: cloning and expression

Complementary DNAs coding for four Desmodus rotundus salivary plasminogen activators (DSPAs) were isolated and characterized. The predicted amino acid sequences display structural features also found in tissue-type plasminogen activator. The largest forms (DSPA alpha 1 and -alpha 2) contain a signal peptide, a finger (F), an epidermal growth factor (EGF), a kringle, and a serine protease domain, whereas DSPA beta and -gamma lack the F and F-EGF domains, respectively. Additional differences between the four forms suggest that distinct genes code for the members of the DSPA family. Transfection of DSPA-encoding cDNAs, placed under the control of the simian virus 40 late promoter, into COS-1 cells resulted in the secretion of highly fibrin-dependent PAs.

Structural Features Mediating Fibrin Selectivity of Vampire Bat Plasminogen Activators

The distinguishing characteristic of vampire bat (Desmodus rotundus) salivary plasminogen activators (DSPAs) is their strict requirement for fibrin as a cofactor. DSPAs consist of structural modules known from urokinase (u-PA) and tissue-type plasminogen activator (t-PA) such as finger (F), epidermal growth factor (E), kringle (K), and protease (P), combining to four genetically and biochemically distinct isoenzymes, exhibiting the formulas FEKP (DSPAα1 and α2) and EKP and KP (DSPAβ and DSPA). Only DSPAα1 and α2 bind to fibrin. All DSPAs are single-chain molecules, displaying substantial amidolytic activity. In a plasminogen activation assay, all four DSPAs are almost inactive in the absence of fibrin but strongly stimulated by fibrin addition. The catalytic efficiency (k/K) of DSPAα1 increases 10-fold, whereas the corresponding value of t-PA is only 550. The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPAα1, α2, β, , and t-PA was found to be 13,000, 6500, 250, 90, and 72, respectively. Whereas all DSPAs are therefore more fibrin dependent and fibrin selective than t-PA, the extent depends on the respective presence of the various domains. The introduction of a plasmin-sensitive cleavage site in a position akin to the one in t-PA partially obliterates fibrin cofactor requirement. Fibrin dependence and fibrin selectivity of DSPAs are accordingly mediated by fibrin binding, which involves the F domain, as yet undefined determinants within the K and P domains, and by the absence of a plasmin-sensitive activation site. These findings transcend the current understanding of fibrin-mediated stimulation of plasminogen activation: in addition to fibrin binding, specific protein-protein interactions come into play, which stabilize the enzyme in its active conformation.

Expression profiling of IL-10-regulated genes in human monocytes and peripheral blood mononuclear cells from psoriatic patients during IL-10 therapy.

Interleukin‐10 (IL‐10), originally identified as an inhibitor of pro‐inflammatory cytokine production, exerts multiple immunomodulatory functions. Its ability to inhibit a Th1 response has been used in clinical trials for the treatment of inflammatory diseases including psoriasis. However, little is known about the molecular mechanisms of IL‐10 functions. We aimed at identifying possiblemediators of in vitro IL‐10 treatment in monocytes by gene chip technology using Hu95a Affymetrix mRNA arrays with 12,000 genes. To prove relevance of the identified genes for the clinicalsituation we compared these in vitro results with genes being regulated by IL‐10 in peripheral blood mononuclear cells from psoriatic patients undergoing IL‐10 therapy. A high proportion of the 1,600 genes up‐regulated and 1,300 genes down‐regulated in vitro was found to be similarly regulated in vivo. Some genes, which were previously unknown to be regulated by IL‐10, can be assigned to known IL‐10 functions like e.g. the increase of pathogen clearance. Other new potentially immunomodulating genes have been identified to be regulated by IL‐10, but their impact needs to be experimentally evaluated. We could confirm a recently reported up‐regulation of heme oxygenase‐1 (HO‐1). However, we demonstrate that the anti‐inflammatory mechanisms of IL‐10 remain functional even when HO‐1 is irreversibly inhibited.

Comparison of Proteomic and Genomic Analyses of the Human Breast Cancer Cell Line T47D and the Antiestrogen-resistant Derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex™, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.

Bovine urokinase-type plasminogen activator and its receptor: cloning and induction by retinoic acid.

Full-length cDNAs encoding bovine urokinase-type plasminogen activator (u-PA) and urokinase receptor (u-PAR) were cloned from an aortic endothelial cell cDNA library using PCR-amplified cDNA fragments as probes. Bovine u-PA amino acid identity ranges from 79.5 to 70.9% when compared to its pig, human, baboon and mouse analogues, while bovine u-PAR is 61.8 and 59.6% identical to its human and mouse counterparts, respectively. All Cys residues previously found in mature u-PA and u-PAR from these different species are also conserved in the bovine molecules. Bovine u-PA and its cell-surface receptor display one and six potential sites of N-linked glycosylation, respectively. Northern blot hybridization demonstrated a moderate induction of u-PA and u-PAR mRNA in bovine aortic endothelial cells after treatment with 10 nM and 1 microM retinoic acid for 8 hours.

cDNA cloning of two closely related forms of human germ cell nuclear factor (GCNF)

Germ cell nuclear factor (GCNF) was initially described in the mouse as a germ cell-specific orphan member of the nuclear receptor superfamily. Two full-length cDNAs encoding variants of the human germ cell nuclear factor (GCNF) differing by only one amino acid residue have now been isolated from a human testis cDNA library and characterised. The encoded proteins show 98.3% and 82.7% amino acid identity with mouse and Xenopus GCNF, respectively. Northern blot hybridisation revealed the expression of an 8 kb human GCNF mRNA exclusively in the testis. The alignment of the GCNF protein sequence with other members of the nuclear hormone receptor family suggests an unusual structural organisation of the C-terminal portion of the putative ligand-binding domain.

IL-10 protects monocytes and macrophages from complement-mediated lysis

Phagocytes, such as monocytes and macrophages, are important cells of the innate immunity in the defense against microbes. So far, it is unclear how these cells survive at the site of combat against microbes, where a hostile inflammatory environment prevails with strong complement activity. We hypothesized that IL‐10, a key cytokine involved in the resolution of inflammation, induces resistance to complement attack. Here, we demonstrate for the first time such a cell‐protective effect of IL‐10 on human monocytes and macrophages. IL‐10 is indeed able to protect these cell types in an in vitro model of complement lysis triggered by an anti‐MHCI antibody or by binding of zymosan. Investigating potential underlying mechanisms, we found that IL‐10 up‐regulated the expression of complement regulatory membrane protein CD59 and the general cell‐protective stress protein HO‐1 in human monocytes. However, further functional analysis failed to link these individual IL‐10‐mediated effects with the increased protection from complement lysis. Blocking the protective effect of CD59 with an antibody increased complement lysis but did not abrogate the IL‐10‐protective effect. Interestingly, chemical interference with HO‐1 activity did abrogate the protective effect of IL‐10, but siRNA‐mediated knockdown of HO‐1 did not confirm this observation. Our results suggest that IL‐10 generates pathogen‐clearing phagocytes, which are resistant to complement lysis and thereby, enabled to survive longer in a hostile inflammatory environment.

The Target Discovery Process

In order to minimise attrition rates in drug development projects, a target discovery process is implemented to select and characterise the most suitable candidate kinase targets, before lead identification and lead optimisation are embarked upon. The process consists of 1) target selection, 2) target assessment, and 3) target validation. This rational approach to target discovery, as a prerequisite for lead discovery, ensures that new therapeutic targets fulfil a set of general criteria, as well as indication‐specific, descriptive and functional ones. The approach should ultimately maximise the likelihood of achieving target‐selective inhibition by small‐molecule inhibitors with minimal in vivo side effects and a therapeutic effect based on a sound biological hypothesis.

High-level secretion of the four salivary plasminogen activators from the vampire bat Desmodus rotundus by stably transfected baby hamster kidney cells.

The cDNAs coding for the four Desmodus rotundus salivary plasminogen activators (DSPAs) were subcloned into the mammalian expression vector, pMPSV/CMV, which carries the myeloproliferative sarcoma virus promoter and the cytomegalovirus enhancer. These constructs were transfected, together with plasmids harbouring Geneticin (G418)-resistance and puromycin-resistance genes, into baby hamster kidney cells. Through the selective pressure of both antibiotics, cell clones constitutively overexpressing the DSPA alpha 1, DSPA alpha 2, DSPA beta or DSPA gamma cDNAs were obtained. Secretion of active DSPAs was confirmed by zymographic analysis and quantified using a fibrin plate assay and ELISA.