Uwe Jerke

The Jak/Stat pathway and urokinase receptor signaling in human aortic vascular smooth muscle cells.

The binding of urokinase plasminogen activator (uPA) to its specific receptor (uPAR) facilitates migration of vascular smooth muscle cells (VSMC). However, the signaling cascade utilized by the urokinase receptor is only incompletely understood. We investigated intracellular uPA/uPAR signaling in human aortic VSMC from the cell membrane to the nucleus. uPA binding to VSMC induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with molecular masses of 53–60, 85–90, and 130–140 kDa. By using co-immunoprecipitation techniques and in vitro kinase assays, the uPAR-associated proteins were identified as Janus (Jak) and Src non-receptor protein-tyrosine kinases (PTK) Jak1, Tyk2, and p59 fyn , p53/56 lyn , p53/59 hck , and p55 fgr . Furthermore, uPA induced a time-dependent reversible translocation of the Stat1 (signal transducer and activator of transcription) protein to the VSMC nuclei, as shown by confocal microscopy studies. Using an electrophoretic mobility shift assay, we then demonstrated that Stat1 is rapidly activated in response to stimulation with uPA and specifically binds to the DNA regulatory elements GAS (interferon-γ activation site) and ISRE (interferon-stimulated response element). Mobility supershift experiments confirmed DNA-protein complexes containing Stat1 protein. Migration experiments with double immunofluorescence staining revealed polarization of uPAR, and colocalization with Jak1 and Tyk2 to the leading edge of the migrating cells. Under the same conditions, Jak2, Jak3, and the Src-PTKs remained randomly distributed over the entire body of the cells. Our studies therefore suggest that, in VSMC, the uPAR-signaling complex utilizes at least two different mechanisms, a direct signaling pathway utilizing the Jak/Stat cascade and a second signal transduction mechanism via Src-like protein-tyrosine kinases. uPA-induced signaling via Jak/Stat is most likely involved in the regulation of cell migration, while the functional purpose of the uPA-associated Src-PTK activation remains to be elucidated.

Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin

BACKGROUND Urokinase (uPA) and the urokinase receptor (uPAR) form a multifunctional system capable of concurrently regulating pericellular proteolysis, cell-surface adhesion, and mitogenesis. The role of uPA and uPAR in directed proteolysis is well established and its function in cellular adhesiveness has recently been clarified by numerous studies. The molecular mechanisms underlying the mitogenic effects of uPA and uPAR are still unclear, however. RESULTS We identified mechanisms that might participate in uPA-related mitogenesis in human vascular smooth muscle cells and demonstrated that uPA induces activation of a unique signaling complex. This complex contains uPAR and two additional proteins, nucleolin and casein kinase 2, which are implicated in cell proliferation. Both proteins were isolated by affinity chromatography on uPA-conjugated cyanogen-bromide-activated Sepharose 4B and were identified using nano-electrospray mass spectrometry and immunoblotting. We used laser scanning and immunoelectron microscopy studies to further demonstrate that nucleolin and casein kinase 2 are located on the cell surface where they colocalize with the uPAR. Moreover, the proteins were co-internalized into the cell as an entire complex. Immunoprecipitation experiments in combination with an in vitro kinase assay demonstrated a specific association of uPAR with nucleolin and casein kinase 2 and revealed a uPA-induced activation of casein kinase 2, which presumably led to phosphorylation of nucleolin. Blockade of nucleolin and casein kinase 2 with specific modulators led to the inhibition of uPA-induced cell proliferation. CONCLUSIONS We conclude that in human vascular smooth muscle cells, uPA induces the formation and activation of a newly identified signaling complex comprising uPAR, nucleolin, and casein kinase 2, that is responsible for the uPA-related mitogenic response. The complex is not a unique feature of vascular smooth muscle cells, as it was also found in other uPAR-expressing cell types.

Complement Receptor Mac-1 Is an Adaptor for NB1 (CD177)-mediated PR3-ANCA Neutrophil Activation

The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1pos/PR3high neutrophils, but not in the mNB1neg/PR3low subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1pos/mPR3high neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when β2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.

Urokinase Induces Activation and Formation of Stat4 and Stat1-Stat2 Complexes in Human Vascular Smooth Muscle Cells

Urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) act in concert to stimulate cytoplasmic signaling machinery and transcription factors responsible for cell migration and proliferation. Recently we demonstrated that uPA activates the Janus kinase/signal transducers and activators of transcription (Stat1) signaling in human vascular smooth muscle and endothelial cells. However, the important question whether other transcription factors of the Stat family, in addition to Stat1, are involved in the uPAR-related signaling has not been addressed. In this study, we demonstrate that Stat4 and Stat2, but not Stat3, Stat5, or Stat6, are rapidly activated in response to uPA. We demonstrate further that Stat4 and Stat2 rapidly and transiently translocate to the cell nucleus where they bind specifically to the regulatory DNA elements. Analysis of Stat complexes formed in response to uPA revealed a Stat2-Stat1 heterodimer, which lacks p48, a DNA-binding protein known to combine with Stat1-Stat2. This new uPA-induced Stat2-Stat1 heterodimer binds to GAS (the interferon-γ activation site) distinct from the interferon-stimulated response element to which the p48 protein containing complexes generally bind. We conclude that uPA activates a specific and unusual subset of latent cytoplasmic transcription factors in human vascular smooth muscle cells that suggests a critical role of uPA in these cells.

Urokinase-dependent Human Vascular Smooth Muscle Cell Adhesion Requires Selective Vitronectin Phosphorylation by Ectoprotein Kinase CK2

Urokinase (uPA)- and urokinase receptor (uPAR)-dependent cell adhesion to the extracellular matrix protein vitronectin (Vn) is an important event in wound healing, tissue remodeling, immune response, and cancer. We recently demonstrated that in human vascular smooth muscle cells (VSMC) uPA/uPAR are functionally associated with the ectoprotein kinase casein kinase-2 (CK2). We now asked whether CK2 regulates uPA-dependent cell adhesion to Vn, since the latter is a natural CK2 substrate. We found that Vn is indeed selectively phosphorylated by CK2 and that this phosphorylation is uPA-regulated in VSMC. Vn induces release of ecto-CK2 from the cell surface via a process termed as “shedding.” CK2-mediated Vn phosphorylation was decisive for the uPA-dependent VSMC adhesion. Specific inhibition of CK2 completely abolished the uPA-induced cell adhesion to Vn. This effect was specific for cell adhesion to Vn and required participation of both uPAR and αvβ3 integrins as adhesion receptors. CK2 localization at the cell surface was highly dynamic; Vn induced formation of clusters where CK2 colocalized with uPAR and αvβ3 integrins. These results indicate that the uPA-dependent VSMC adhesion is a function of selective Vn phosphorylation by the ectoprotein kinase CK2 and suggest a regulatory role for Vn phosphorylation in the uPA-directed adhesive process.

β2 Integrin-mediated Cell-Cell Contact Transfers Active Myeloperoxidase from Neutrophils to Endothelial Cells

Background: How endothelial cells (ECs) could acquire exogenous neutrophil myeloperoxidase (MPO) is unknown. Results: ECs acquired enzymatically active MPO directly from neutrophils, via β2 integrin-mediated cell-cell contact independent of extracellular MPO release. Conclusion: Neutrophils directly transfer MPO to ECs by cell-cell contact. Significance: Direct delivery of MPO to ECs may contribute to the vascular pathology and dysfunction seen in atherosclerosis and vasculitides. Atherosclerosis and vasculitis both feature inflammation mediated by neutrophil-endothelial cell (EC) contact. Neutrophil myeloperoxidase (MPO) can disrupt normal EC function, although the mechanism(s) by which MPO is transferred to ECs are unknown. We tested the hypothesis that close, β2 integrin-dependent neutrophil-EC contact mediates MPO transfer from neutrophils to ECs. We used sensitive MPO assays and flow cytometry to detect MPO in ECs and demonstrate that ECs acquired MPO when contacted by neutrophils directly but not when ECs and neutrophils were separated in Transwells. The transfer was dependent on neutrophil number, exposure time, and incubation temperature. Transfer occurred in several EC types, increased with endotoxin, was not accompanied by MPO release into the medium, and was not abrogated by inhibiting degranulation to secretagogues. Confocal microscopy showed MPO internalization by ECs with cytoplasmic and nuclear staining. Neutrophils and ECs formed intimate contact sites demonstrated by electron microscopy. Blocking CD11b or CD18 β2 integrin chains, or using neutrophils from CD11b gene-deleted mice, reduced MPO transfer. EC-acquired MPO was enzymatically active, as demonstrated by its ability to oxidize the fluorescent probe aminophenyl fluorescein in the presence of a hydrogen peroxide source. The data suggest an alternative to EC uptake of soluble MPO, namely the cell contact-dependent, β2 integrin-mediated transfer from neutrophils. The findings could be of therapeutic relevance in atherosclerosis and vasculitis.

Neutrophil serine proteases exert proteolytic activity on endothelial cells

Neutrophil serine proteases (NSPs) are released from activated neutrophils during inflammation. Here we studied the transfer of the three major NSPs, namely proteinase 3, human neutrophil elastase, and cathepsin G, from neutrophils to endothelial cells and used an unbiased approach to identify novel endothelial NSP substrates. Enzymatically active NSPs were released from stimulated neutrophils and internalized by endothelial cells in a dose- and time-dependent manner as shown by immunoblotting, flow cytometry, and the Boc-Ala substrate assay. Using terminal-amine isotopic labeling of substrates in endothelial cells, we identified 121 peptides from 82 different proteins consisting of 36 substrates for proteinase 3, 30 for neutrophil elastase, and 28 for cathepsin G, respectively. We characterized the extended cleavage pattern and provide corresponding IceLogos. Gene ontology analysis showed significant cytoskeletal substrate enrichment and confirmed several cytoskeletal protein substrates by immunoblotting. Finally, ANCA-stimulated neutrophils released all three active NSPs into the supernatant. Supernatants increased endothelial albumin flux and disturbed the endothelial cell cytoskeletal architecture. Serine protease inhibition abrogated this effect. Longer exposure to NSPs reduced endothelial cell viability and increased apoptosis. Thus, we identified novel NSP substrates and suggest NSP inhibition as a therapeutic measure to inhibit neutrophil-mediated inflammatory vascular diseases.

Stat1 Nuclear Translocation by Nucleolin upon Monocyte Differentiation

Background Members of the signal transducer and activator of transcription (Stat) family of transcription factors traverse the nuclear membrane through a specialized structure, called the nuclear pore complex (NPC), which represents a selective filter for the import of proteins. Karyophilic molecules can bind directly to a subset of proteins of the NPC, collectively called nucleoporins. Alternatively, the transport is mediated via a carrier molecule belonging to the importin/karyopherin superfamily, which transmits the import into the nucleus through the NPC. Methodology/Principal Findings In this study, we provide evidence for an alternative Stat1 nuclear import mechanism, which is mediated by the shuttle protein nucleolin. We observed Stat1-nucleolin association, nuclear translocation and specific binding to the regulatory DNA element GAS. Using expression of nucleolin transgenes, we found that the nuclear localization signal (NLS) of nucleolin is responsible for Stat1 nuclear translocation. We show that this mechanism is utilized upon differentiation of myeloid cells and is specific for the differentiation step from monocytes to macrophages. Conclusions/Significance Our data add the nucleolin-Stat1 complex as a novel functional partner for the cell differentiation program, which is uniquely poised to regulate the transcription machinery via Stat1 and nuclear metabolism via nucleolin.

Characterization of the CD177 interaction with the ANCA antigen proteinase 3

Proteinase 3 is a serine protease found in neutrophil granules and on the extracellular neutrophil membrane (mPR3). mPR3 is a major antigen for anti-neutrophil cytoplasmic antibodies (PR3-ANCAs), autoantibodies causing fatal autoimmune diseases. In most individuals, a subpopulation of neutrophils also produce CD177, proposed to present additional PR3 on the surface, resulting in CD177neg/mPR3low and CD177pos/mPR3high neutrophil subsets. A positive correlation has been shown between mPR3 abundance, disease incidence, and clinical outcome. We present here a detailed investigation of the PR3:CD177 complex, verifying the interaction, demonstrating the effect of binding on PR3 proteolytic activity and explaining the accessibility of major PR3-ANCA epitopes. We observed high affinity PR3:CD177 complex formation by surface plasmon resonance. Using flow cytometry and a PR3-specific FRET assay, we found that CD177 binding reduced the proteolytic activity of PR3 in vitro using purified proteins, in neutrophil degranulation supernatants containing wtPR3 and directly on mPR3high neutrophils and PR3-loaded HEK cells. Finally, CD177pos/mPR3high neutrophils showed no migration advantage in vitro or in vivo when migrating from the blood into the oral cavity. We illuminate details of the PR3:CD177 interaction explaining mPR3 membrane orientation and proteolytic activity with relevance to ANCA activation of the distinct mPR3 neutrophil populations.

Lessons from a double‐transgenic neutrophil approach to induce antiproteinase 3 antibody–mediated vasculitis in mice

ANCA to either PR3 or MPO are found in patients with necrotizing vasculitis and glomerulonephritis. ANCA binding to their target antigens on neutrophils and subsequent neutrophil activation are pivotal disease mechanisms that lead to vascular inflammation and necrosis. ANCA interaction with PR3 is more complex than with MPO as the neutrophil‐specific CD177 receptor is involved in PR3 surface expression and PR3‐ANCA–induced neutrophil activation. Modeling human disease is important to clinical research. Highly successful mouse models of MPO‐ANCA vasculitis exist; however, recapitulating PR3‐ANCA vasculitis has not been successful. We generated double‐transgenic (DT) mice that expressed human PR3 and CD177 under a myeloid‐specific huMRP8 promoter in an attempt to model PR3‐ANCA vasculitis. DT mice strongly expressed the human transgenes in and on murine neutrophils and bound murine and human anti‐PR3 antibodies. Nevertheless, passive transfer of these antibodies into LPS‐primed DT mice or immunization of C57BL/6 mice with human PR3 followed by irradiation and transplantation of DT bone marrow failed to induce glomerulonephritis. Further analyses revealed that anti‐PR3 antibodies did not activate DT neutrophils as shown by superoxide generation. Moreover, we found that mice did not properly process human pro‐PR3 into mature PR3 and, consequently, the signaling complex between PR3, CD177, and CD11b, which promotes neutrophil activation by anti‐PR3 antibodies, failed to form. We conclude that important species differences in PR3 and CD177 exist between men and mice that prevented successful generation of a murine anti‐PR3 antibody model.