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,2,2
* Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, Homburg/Saar, Germany;
Clinic of Operative Dentistry and Periodontology, University of Saarland Hospital, Homburg/Saar, Germany;
Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany;
Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany,
|| Institute of Medical Microbiology, University Hospital, Münster, Germany,
¶ Department of Medical Biochemistry and Molecular Biology, University of Saarland Hospital, Homburg/Saar, Germany; and
# Department of Dermatology, Heinrich-Heine-University, Düsseldorf, Germany
1Correspondence: Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, D-66421 Homburg/Saar, Germany. E-mail: astrid.sobke{at}gmx.de
SPECIFIC AIMS
The secreted extracellular adherence protein (Eap) from Staphylococcus aureus was previously reported to severely impair neutrophil recruitment, repair angiogenesis, and wound healing in S. aureus-infected mice. Because the broad spectrum of Eap ligands includes angiogenic costimulators like fibrinogen, ICAM-1, and a range of extracellular matrix (ECM) proteins, the aim of this study was to decipher the influence of this versatile adhesin on molecular signaling events and the angiogenic behavior of endothelial cells.
PRINCIPAL FINDINGS
1. Eap inhibits the angiogenic response of primary endothelial cells
Endothelial activation by angiogenic growth factors results in transcriptional up-regulation of genes like the "decay- accelerating factor" and "tissue factor" (TF) as an early event. Here, we found mRNA levels of DAF to be raised approximately twofold by either vascular endothelial growth factor (VEGF)165 or basic fibroblast growth factor (bFGF) in HUVEC, whereas the expression of TF was increased approximately fivefold by VEGF165 (Fig. 1
A). Cotreatment of HUVEC with 20 µg/ml of purified Eap resulted in a strongly attenuated response, the induction being reduced about half. Endothelial proliferation, as assessed by measuring de novo DNA synthesis (Fig. 1B
), as well as by cell number counts, was even more severely impaired by Eap: [3H]thymidine incorporation was raised 4.0 ± 1.6 and 8.7 ± 2.7 fold in HUVEC by VEGF165 and bFGF, respectively. In the presence of 20 µg/ml Eap, this mitogenic response was completely abrogated (1.28±0.66 and 1.36±0.17). The antiproliferative activity of Eap was concentration-dependent, with complete inhibition seen at 20 µg/ml. Similarly, the bFGF- and VEGF-induced capillary-like sprout formation of bovine retinal endothelial cells (BREC) grown in a fibrin 3D matrix was efficiently repressed by the inclusion of as little as 5 µg/ml Eap (Fig. 1C
).
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2. Eap inhibits VEGF165 and bFGF-induced ERK phosphorylation but has variable effects on Akt phosphorylation
We next immunoblotted lysates of stimulated HUVEC with phosphospecific antibodies directed against the activated forms of key kinases in angiogenic transduction, i.e. ERK1/2 (phospho-Thr202/Tyr204), p38 (phospho-Thr180/Tyr182), and Akt/PKB (phospho-Ser-473). Preincubation with Eap led to a significant reduction in the level of the subsequent VEGF165-triggered ERK1/2 phosphorylation without altering the kinetics of the response (Fig. 2
A). Under the same conditions, Eap did not affect the VEGF165-induced p38 and Akt/PKB phosphorylation. This specific suppression of the MAPK pathway through Eap was not restricted to the VEGF165 elicited response, as similar results were obtained in bFGF- stimulated HUVEC (Fig. 2B
). In contrast, there was an augmental effect of Eap on the bFGF-induced Akt/PKB phosphorylation; while bFGF on its own caused only a marginal increase in Akt/PKB phosphorylation at serine 473, in the presence of Eap, this was clearly enhanced. Similarly, there was also a minor increase of p38 phosphorylation in the presence of Eap. The observed effects of Eap were again concentration-dependent. Comparable results were also seen in BREC, except that these microvascular cells responded to bFGF with a strong phosphorylation of Akt/PKB, which was actually inhibited by Eap.
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3. Inhibition of the MAPK cascade occurs upstream of ERK1/2 and downstream of PLC
To delineate the target of Eap in more detail, we studied the effects of combinations of Eap, the PKC inhibitor GF109203X, and the farnesyl transferase inhibitor manumycin A (indirect inhibitor of Ras) on the VEGF-induced MAPK cascade. Both GF109203X and Eap had a substantial inhibitory effect on ERK1/2 phosphorylation, with the combined effect resulting in the complete blockage of the phosphorylation response. In comparison, manumycin A treatment on its own had a weaker impact on ERK1/2 phosphorylation, whereas concomitant treatment with Eap resulted again in an additive effect. To test whether the impact of Eap was independent of receptor activation, we examined the effects of Eap, GF109203X, and manumycin A on activation of the MAPK pathway during stimulation with the phorbol ester TPA. The findings were comparable to those seen on VEGF stimulation. Immunoblotting with a phospho-Ser-217/221 specific antibody revealed Eap to have similar effects on MEK activation as on ERK. Activation of PKC requires its translocation to the plasma membrane and autophosphorylation. Eap had no impact on the membrane translocation or phosphorylation levels of PKC
.
4. Eap exerts its effect at the level of Ras but not at the level of Raf-1
Raf-1 constitutes an important relay point within the MAPK cascade, integrating positive and negative inputs from upstream pathways. Activation of Raf-1 is accompanied by a characteristic shift in electophoretic mobility due to an increased phosphorylation state.
Thus, stimulation of HUVEC with TPA induced the occurrence of an additional, retarded band on Raf-1 immunoblots. The intensity of this band was again reduced on treatment with Eap. Pull-down assays were used to investigate Ras activation. There was a profound reduction in activated, GTP-bound Ras after VEGF, as well as bFGF stimulation in Eap-exposed HUVEC, indicating interference of Ras activation as the earliest target of Eap blockage of MAPK pathway activation.
CONCLUSIONS AND SIGNIFICANCE
Staphylococcus aureus is the "classical" and most common causative agent of abscess formation and surgical site infections (SSI), which typically are accompanied by impaired wound healing and defects in granulationtissue formation. Previously, we reported Eap to severely impair angiogenesis and wound healing in S. aureus-infected mice. However, since Eap was shown to have potent immunosuppressive activities, it was so far unclear to what extent its adverse effects on angiogenesis are the consequence of interference with the inflammatory response. Examining isolated primary EC, we here demonstrate that Eap directly blocks the angiogenic response at different stages of activation (transcriptional up-regulation, proliferation, differentiation into tubular networks) and in EC of variable origin (microvascular bovine retinal vs. macrovascular human umbilical vein EC). At the molecular level, these effects are shown to be based on corresponding alterations in proangiogenic signal transduction, in particular, ERK1/2 phosphorylation was found to be severely disturbed in the presence of Eap. In former studies staphylococcal surface proteins were reported to bind basic growth factors like bFGF in a charge-dependent manner and interference with VEGF and bFGF binding to endothelial cells is also part of the antiangiogenic activity of thrombospondins. By demonstrating comparable effects of Eap on TPA-induced ERK1/2 phosphorylation, we can exclude such competition for binding sites as the basis of the effect described here and show that inhibition has to occur downstream of tyrosine kinase receptor activation. Similar to endogenous inhibitors of angiogenesis, like the 16-kDa human prolactin fragment (16 K hPRL) or angiopoietin-1 (Ang-1), Eap was found to interfere with ERK1/2 activation by blocking Ras-GTP formation. In bFGF-stimulated BREC, Eap also caused the complete blockage of the strong Akt phosphorylation observed in these cells. Because PI3-kinase induction upstream of Akt activation may occur in a Ras-dependent or -independent manner, this implies the involvement of Ras in this cell type. Contrary to its effect on Ras-dependent pathways, Eap had a positive influence on basal p38 and Akt activation levels, and there was also a synergistic effect with bFGF on phospho-Ser-473- Akt formation in HUVEC (2.5-fold increase with Eap over bFGF alone). Akt phosphorylation at serine 473 leads to subsequent inhibition of proapoptotic factors and nuclear exclusion of the "forkhead box class-O winged helix transcription factor" (FoxO). This was previously reported as an important mechanism through which Ang-1 modulates endothelial function. An enhancement of Akt activation may thus add to Eap’s antiangiogenic activity. Further studies will be needed to determine the exact nature of the Eap receptor(s). However, during recent years, it has become increasingly clear that endothelial responses to bFGF/VEGF are not solely mediated by their specific tyrosine kinase receptors but rather require the sequestration of a number of coreceptors together with FGF receptor 1 (FGFR-1) and VEGF receptor 2 (VEGFR-2/ KDR/Flk-1) at specific membrane subdomains. Elucidation of Eap’s interaction with the endothelium may thus help to shed some light into these complex processes. The potent anti-inflammatory and antiangiogenic activities of Eap make this versatile protein not only an important virulence factor during S. aureus infection but open new perspectives for future pharmacological application, for instance in inhibiting pathological neovascularization as occurs in tumor growth, or curbing the overshooting host responses in sepsis.
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FOOTNOTES
2 Present address: Experimental Immunology Branch, NCI, Bethesda, MD, USA. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5764fje
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